US11624077B2 - Gene knockout method - Google Patents

Gene knockout method Download PDF

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US11624077B2
US11624077B2 US16/637,591 US201716637591A US11624077B2 US 11624077 B2 US11624077 B2 US 11624077B2 US 201716637591 A US201716637591 A US 201716637591A US 11624077 B2 US11624077 B2 US 11624077B2
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sequence
donor
sgrna
donor dna
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US20200255866A1 (en
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Wensheng Wei
Yiou Chen
Yuexin ZHOU
Hongmin Zhang
Pengfei YUAN
Yuan Liu
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Peking University
Edigene Biotechnology Inc
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3517—Marker; Tag

Definitions

  • the present invention relates to the genome editing technology, in particular to a gene knockout method.
  • ZFNs zinc finger nucleases
  • TALENs transcription activator-like effector nucleases
  • CRISPR/Cas9 systems employ different mechanisms to generate sequence-specific double-strand breaks (DSBs) and subsequently trigger natural repair systems to complete sequence-specific modifications [8, 9].
  • DLBs sequence-specific double-strand breaks
  • CRISPR/Cas9 system has become particularly popular for its efficiency and ease of operation.
  • the CRISPR/Cas9 system was originally used by the bacterial immune system to fight against foreign viruses or plasmids.
  • a Cas9 endonuclease cleaves a double-stranded DNA under the guidance of an sgRNA, resulting in a double-strand break in the genome and production of repair errors (base deletions or insertions) by making use of the instability of the cell genome repair, thereby achieving the effect of genome editing.
  • the CRISPR/Cas9 system has unprecedented advantages in design- and sequence-specificity-based genomic researches, the genome editing triggered by the CRISPR/Cas9 system is still a rare event in a cell population. It requires tedious labour to get real genetically edited single clones. Therefore, the system is still technically challenging, even for a simple task of producing a gene knockout in a mammalian cell [15].
  • the present invention provides a donor construct and a gene knockout method, as well as a system and kit for the gene knockout.
  • the gene knockout method of the present invention uses a marker gene comprised in the donor construct to enrich cells in which a gene is knocked out, thereby improving the efficiency of a sequence-specific nuclease generated gene knockout.
  • a donor construct is provided, wherein the donor construct is a linear donor DNA or is capable of been cleaved in a cell to produce a linear donor DNA, and the linear donor DNA sequentially comprises, from the middle to both ends: an expression cassette; a short sequence extension comprising a reverse termination codon located at the 5′-end of the expression cassette and a short sequence extension consisting of a forward termination codon located at the 3′-end of the expression cassette; a target sequence located at the 5′-end and/or 3′-end, comprising a target site cleavable by the sequence-specific nuclease; and protective sequences located at both ends; wherein the expression cassette comprises a promoter-driven marker gene.
  • the linear donor DNA is a double-stranded linear donor DNA.
  • the donor construct is a linear donor DNA.
  • sequence-specific nuclease is a zinc finger nuclease (ZFN).
  • sequence-specific nuclease is a transcription activator-like effector nuclease (TALEN).
  • TALEN transcription activator-like effector nuclease
  • sequence-specific nuclease is a Cas9 nuclease.
  • sequence-specific nuclease is an NgAgo nuclease.
  • the linear donor DNA only has a target sequence at the 5′-end or the 3′-end.
  • the linear donor DNA has target sequences at both ends, respectively.
  • the target sequences at both ends of the linear donor DNA are the same.
  • the target sequences at both ends of the linear donor DNA are different. In a further embodiment, the different target sequences at both ends of the linear donor DNA are derived from the same gene. In another further embodiment, the different target sequences at both ends of the linear donor DNA are derived from different genes.
  • the marker gene is an antibiotic resistance gene or a fluorescent protein gene.
  • the protective sequence is 5-30 bp, most preferably 20 bp, in length.
  • a method for generating a gene knockout in a cell comprising the steps of:
  • the donor construct is a linear donor DNA or is capable of been cleaved in a cell to produce a linear donor DNA
  • the linear donor DNA sequentially comprises, from the middle to both ends: an expression cassette; a short sequence extension consisting of a reverse termination codon located at the 5′-end of the expression cassette and a short sequence extension consisting of a forward termination codon located at the 3′-end of the expression cassette; a target sequence located at the 5′-end and/or 3′-end, comprising a target sequence cleavable by the sequence-specific nuclease; and protective sequences located at both ends; and the expression cassette comprises a promoter-driven marker gene;
  • linear donor DNA is inserted into the specific target site in the cell genome by non-homologous end joining
  • the linear donor DNA is a double-stranded linear donor DNA.
  • the donor construct is a linear donor DNA.
  • the linear donor DNA only has a target sequence at the 5′-end or the 3′-end.
  • the linear donor DNA has target sequences at both ends, respectively.
  • the target sequences at both ends of the linear donor DNA are the same.
  • the target sequences at both ends of the linear donor DNA are different. In a further embodiment, the different target sequences at both ends of the linear donor DNA are derived from the same gene. In another further embodiment, the different target sequences at both ends of the linear donor DNA are derived from different genes.
  • sequence-specific nuclease is a zinc finger nuclease (ZFN).
  • sequence-specific nuclease is a transcription activator-like effector nuclease (TALEN).
  • TALEN transcription activator-like effector nuclease
  • sequence-specific nuclease is a Cas9 nuclease.
  • the method further comprises introducing into the cell a guide RNA (gRNA) that recognizes a specific target site in the cell genome, wherein the target sequence in the linear donor DNA is recognized by the gRNA.
  • gRNA guide RNA
  • the gRNA is an sgRNA.
  • the method further comprises introducing into the cell an sgRNA that recognizes a single specific target site in the cell genome, wherein the target sequence comprising the target site recognized by the sgRNA is located at the 5′-end and/or the 3′-end of the linear donor DNA.
  • the target sequence comprising the target site recognized by the sgRNA is derived from a single gene in the cell genome.
  • the target sequence comprising the target site recognized by the sgRNA is a consensus sequence of two or more genes in the cell genome, provided that the consensus sequence has no more than one base difference from the sequences in any of the two or more genes at positions corresponding to the consensus sequence.
  • the method further comprises introducing into the cell two sgRNAs that recognize two specific target sites in one gene in the cell genome, wherein two target sequences respectively comprising the two target sites recognized by the two sgRNAs are located in two linear donor DNAs, respectively, or located at both ends of the same linear donor DNA, respectively.
  • the method further comprises introducing into the cell two or more sgRNAs that recognize two or more specific target sites in the cell genome, wherein two or more target sequences respectively comprising the two or more target sites recognized by the two or more sgRNAs are located at both ends of the same linear donor DNA, respectively, or located in different linear donor DNAs.
  • the two or more specific target sites in the cell genome are located in different genes, respectively.
  • sequence-specific nuclease is an NgAgo nuclease.
  • the method further comprises introducing into the cell a guide DNA (gDNA) that recognizes a specific target site in the cell genome, wherein the target sequence in the linear donor DNA comprises a target site recognized by the gDNA.
  • gDNA guide DNA
  • the gene knockout can be a single gene knockout or a multi-gene knockout.
  • the multi-gene knockout is a knockout of two or more genes, such as a knockout of three, four, five or more genes.
  • the marker gene is an antibiotic resistance gene or a fluorescent protein gene.
  • the cells are screened by the drug resistance.
  • the cells are screened by the FACS method.
  • the protective sequence is 5-30 bp, most preferably 20 bp, in length.
  • a system or kit for the gene knockout comprising: a sequence-specific nuclease capable of cleaving a specific target site in the cell genome, and a donor construct;
  • the donor construct is a linear donor DNA or is capable of been cleaved in a cell to produce a linear donor DNA
  • the linear donor DNA sequentially comprises, from the middle to both ends: an expression cassette; a short sequence extension consisting of a reverse termination codon located at the 5′-end of the expression cassette and a short sequence extension consisting of a forward termination codon located at the 3′-end of the expression cassette; a target sequence located at the 5′-end and/or 3′-end, comprising a target sequence cleavable by the sequence-specific nuclease; and protective sequences located at both ends; and the expression cassette comprises a promoter-driven marker gene.
  • the linear donor DNA is a double-stranded linear donor DNA.
  • the donor construct is a linear donor DNA. In some other embodiments, the donor construct is a circular donor construct that can be cleaved in a cell to produce a linear donor DNA.
  • sequence-specific nuclease is a zinc finger nuclease (ZFN).
  • sequence-specific nuclease is a transcription activator-like effector nuclease (TALEN).
  • TALEN transcription activator-like effector nuclease
  • sequence-specific nuclease is a Cas9 nuclease.
  • system or kit further comprises an sgRNA that recognizes a specific target site in the cell genome, wherein the target sequence in the linear donor DNA comprises a target site recognized by the sgRNA.
  • the gRNA is an sgRNA.
  • sequence-specific nuclease is an NgAgo nuclease.
  • system or kit further comprises a gDNA that recognizes a specific target site in the cell genome, wherein the target sequence in the linear donor DNA comprises a target site recognized by the gDNA.
  • the marker gene is an antibiotic resistance gene or a fluorescent protein gene.
  • the protective sequence is 5-30 bp, most preferably 20 bp, in length.
  • the cleavage is to generate double-strand breaks (DSBs).
  • a universal donor construct is provided, wherein the universal donor construct is a linear donor DNA or is capable of been cleaved in a cell to produce a linear donor DNA, and the linear donor DNA sequentially comprises, from the middle to both ends: an expression cassette; a short sequence extension consisting of a reverse termination codon located at the 5′-end of the expression cassette and a short sequence extension consisting of a forward termination codon located at the 3′-end of the expression cassette; a universal target sequence located at the 5′-end and/or 3′-end, comprising a target site cleavable by a Cas9 nuclease; and protective sequences located at both ends;
  • the expression cassette comprises a promoter-driven marker gene
  • the universal target sequence is absent in a cell genome to be subjected to a gene knockout.
  • the universal donor construct is a linear donor DNA.
  • the linear donor DNA is a double-stranded linear donor DNA.
  • the linear donor DNA only has the universal target sequence at the 5′-end or the 3′-end.
  • the linear donor DNA has the universal target sequences at both ends, respectively.
  • the marker gene is an antibiotic resistance gene or a fluorescent protein gene.
  • the protective sequence is 5-30 bp, most preferably 20 bp, in length.
  • the universal target sequence in the universal donor construct comprises 5′-GTACGGGGCGATCATCCACA-3′ (SEQ ID NO:1) or 5′-AATCGACTCGAACTTCGTGT-3′ (SEQ ID NO:2).
  • a method for generating a gene knockout in a cell comprising the steps of:
  • a universal donor construct wherein the universal donor construct is a linear donor DNA or is capable of been cleaved in a cell to produce a linear donor DNA, and the linear donor DNA sequentially comprises, from the middle to both ends: an expression cassette; a short sequence extension consisting of a reverse termination codon located at the 5′-end of the expression cassette and a short sequence extension consisting of a forward termination codon located at the 3′-end of the expression cassette; a universal target sequence located at the 5′-end and/or 3′-end, comprising a target site cleavable by a Cas9 nuclease; and protective sequences located at both ends;
  • the expression cassette comprises a promoter-driven marker gene
  • the universal target sequence is absent in the cell genome to be subjected to a gene knockout
  • the donor construct is a linear donor DNA.
  • the linear donor DNA is a double-stranded linear donor DNA.
  • the linear donor DNA only has the universal target sequence at the 5′-end or the 3′-end.
  • the linear donor DNA has the universal target sequences at both ends.
  • the gRNA that recognizes the specific target site in the cell genome may be a gRNA, or a plurality of gRNAs that recognize different target sites in the cell genome, such as two, three, or more gRNAs that recognize different target sites in the cell genome.
  • the different target sites may be located in the same gene or may be located in different genes. When the different target sites are located in different genes respectively, the knockout of multiple genes can be achieved.
  • the gene knockout can be a single gene knockout or a multi-gene knockout.
  • the multi-gene knockout is a knockout of two or more genes, such as a knockout of three, four, five or more genes.
  • the gRNA that recognizes the specific target site in the cell genome is an sgRNA.
  • the gRNA that recognizes the universal target sequence contained in the linear donor DNA is an sgRNA.
  • the sgRNA that recognizes the specific target site in the cell genome and the sgRNA that recognizes the universal target sequence contained in the linear donor DNA are located in the same vector.
  • the sgRNA that recognizes the specific target site in the cell genome and the sgRNA that recognizes the universal target sequence contained in the linear donor DNA are located in different vectors.
  • the marker gene is an antibiotic resistance gene or a fluorescent protein gene.
  • the cells are screened by the drug resistance.
  • the cells are screened by the FACS method.
  • the protective sequence is 5-30 bp, most preferably 20 bp, in length.
  • the universal target sequence in the universal donor construct comprises 5′-GTACGGGGCGATCATCCACA-3′ (SEQ ID NO:1) or 5′-AATCGACTCGAACTTCGTGT-3′ (SEQ ID NO:2).
  • a system or kit for a gene knockout comprising:
  • a universal donor construct wherein the universal donor construct is a linear donor DNA or is capable of been cleaved in a cell to produce a linear donor DNA, and the linear donor DNA sequentially comprises, from the middle to both ends: an expression cassette; a short sequence extension consisting of a reverse termination codon located at the 5′-end of the expression cassette and a short sequence extension consisting of a forward termination codon located at the 3′-end of the expression cassette; a universal target sequence located at the 5′-end and/or 3′-end, comprising a target site cleavable by a Cas9 nuclease; and protective sequences located at both ends;
  • the expression cassette comprises a promoter-driven marker gene
  • the universal target sequence is absent in the cell genome to be subjected to a gene knockout
  • the linear donor DNA is a double-stranded linear donor DNA.
  • the donor construct is a linear donor DNA.
  • the donor construct is a circular donor construct that can be cleaved in a cell to produce a linear donor DNA.
  • the gRNA that recognizes the specific target sites in the cell genome may be a gRNA, or a plurality of gRNAs that recognize different target sites in the cell genome, such as two, three, or more gRNAs that recognize different target sites in the cell genome.
  • the different target sites may be located in the same gene or may be located in different genes. When the different target sites are located in different genes respectively, the knockout of multiple genes can be achieved.
  • the gRNA that recognizes the specific target sequence in the cell genome is an sgRNA.
  • the gRNA that recognizes the universal target sequence contained in the linear donor DNA is an sgRNA.
  • the gRNA that recognizes the specific target site in the cell genome and the gRNA that recognizes the universal target sequence contained in the linear donor DNA are located in the same vector.
  • the gRNA that recognizes the specific target site in the cell genome and the gRNA that recognizes the universal target sequence contained in the linear donor DNA are located in different vectors.
  • the marker gene is an antibiotic resistance gene or a fluorescent protein gene.
  • the protective sequence is 5-30 bp, most preferably 20 bp, in length.
  • the target sequence in the universal donor construct comprises 5′-GTACGGGGCGATCATCCACA-3′ (SEQ ID NO:1) or 5′-AATCGACTCGAACTTCGTGT-3′ (SEQ ID NO:2).
  • FIG. 1 C Comparison of ANTXR1 knockout rates of HeLa cells transfected with sgRNA/pgRNA (with (a dark-colored bar) or without (a light-colored bar) the corresponding donors).
  • the ANTXR1 knockout rate is expressed as the percentage of cells resistant to PA/LFnDTA.
  • FIG. 1 D Summary of ANTXR1 knockout cells enriched when using different gRNAs and their donors.
  • FIGS. 2 A- 2 B show the experimental verification of enrichment of ANTXR1 knockout cells in a pooled population and single clones by donor-mediated puromycin resistance selection.
  • FIG. 2 A Images of different HeLa cell groups treated with or without PA/LFnDTA. Mixed cells were obtained by transfection using sgRNA or pgRNA with or without the corresponding linear donors. The plotting scale is 200 ⁇ m.
  • FIG. 2 B PCR verification of the linear donor-integrated ANTXR1 locus of puromycin-resistant (puro+) single clones.
  • FIGS. 4 A- 4 C show the donor design and experimental verification of enrichment of HBEGF disruption events in HEK293T cells by EGFP.
  • FIG. 4 A Design of a donor targeting the HBEGF gene.
  • FIG. 4 B Images of different HEK293T cell groups treated with or without DT (40 ng/ml). Pooled cells were obtained by transfection using sgRNA (sgRNA2HBEGF) (with or without its corresponding linear donor (Donor HBEGF-sg2 )). The plotting scale is 200 ⁇ m.
  • sgRNA2HBEGF sgRNA2HBEGF
  • Donor HBEGF-sg2 linear donor
  • FIGS. 5 A- 5 F show the donor design and experimental verification of enrichment of ANTXR1 disruption events in HeLa oc cells by puromycin selection.
  • FIG. 5 A Design of a donor targeting ANTXR1. The donor comprises an sgRNA cleavage sequence at the 5′-end (Donor ANTR1-sg1 or Donor ANTXR1-sg2 ) or two gRNAs at both ends (Donor ANTXR1-pg ).
  • FIG. 5 B MTT staining of puromycin-resistant clones of cells transfected with a donor (with or without a gRNA).
  • FIG. 5 C Images of different HeLa oc cell groups treated with or without PA/LFnDTA.
  • FIG. 5 E PCR verification of the linear donor-integrated ANTXR1 locus of puromycin-resistant single clones.
  • FIG. 5 F Summary of ANTXR1 knockout cells enriched when using different gRNAs and their donors.
  • FIGS. 6 A- 6 B show the off-target assessment of a donor insertion in HeLa oc cells by splinkerette PCR (spPCR) analysis.
  • FIG. 6 A The adaptor and primer design for the spPCR analysis.
  • Splink1 and Splink2 primers are complementary to the adaptor sequence, and primers R1 and R2 are complementary to the linear donor sequence.
  • FIG. 6 B The spPCR reaction results.
  • FIGS. 7 A- 7 D show the donor design and experimental verification of enrichment of HBEGF disruption events in HeLa oc cells by puromycin selection.
  • FIG. 7 A Design of a donor targeting HBEGF. The donor comprises an sgRNA cleavage sequence at the 5′-end (Donor HBEGF-sg1 or Donor HBEGF-sg2 ) or two gRNAs at both ends (Donor HBEGF-pg )
  • FIG. 7 B MTT staining of puromycin-resistant clones of cells transfected with a donor (with or without sgRNA/pgRNA).
  • FIG. 7 C PCR verification of the linear donor-integrated HBEGF locus of a puromycin-resistant single clone.
  • FIG. 7 D Summary of HBEGF knockout cells enriched when using different gRNAs and their donors.
  • FIGS. 8 A- 8 F show the donor design and experimental verification of generation of two or more gene knockouts in one step in HeLa oc cells.
  • FIG. 8 A A schematic diagram of NHEJ-based knockin of a linear donor at an sgRNA- or pgRNA-targeting site in PSEN1 and PSEN2 genes.
  • FIG. 8 B- 8 C The sequencing analysis of partial encoding sequences of PSEN1 and PSEN2 in genome, comprising an sgRNA encoding region (underlined) and a mutant allele.
  • Clone 1 ( FIG. 8 B ) was derived from HeLa oc cells transfected with pgRNA PsEN1+PsEN2 /Donorp PSEN1+PSEN2 Clone 2 ( FIG.
  • FIG. 8 C Multiple sequence alignment analysis of the HSPA gene family showing the consensus sequence; an sgRNA targeting the consensus sequence of five HSPA family genes; and the design of the universal linear donor (Donor HSPA ) for enrichment of cells containing multi-gene mutations.
  • the black shaded nucleotides represent the consensus sequence of all the five HSPA genes. Dark gray shaded nucleotides represent the consensus sequence of three or four HSPA genes, while light gray shaded nucleotides represent non-consensus nucleotides.
  • FIG. 8 F Partial encoding sequences of HSPA1A, HSPA1B, HSPA1L and HSPA6 genes in the genome of HeLa clone 3 comprising an sgRNA-targeting region (underlined). Clone 3 was derived from HeLa oc cells transfected with sgRNA HHSPA /Donor HSPA . The shaded nucleotides represent the PAM sequence, and the dashed lines represent deletions. Light gray arrows in the background indicate the direction of the CMV promoter in the donor.
  • FIGS. 9 A- 9 C show the efficiency evaluation of PSEN1 and PSEN2 sgRNAs in HeLa oc cells and the single clone recognition.
  • FIG. 9 B MTT staining of puromycin-resistant clones of cells transfected with a donor (with or without pgRNA PSEN ).
  • FIG. 9 C PCR results of the two linear donor-integrated PSEN1 (L3/R3) and PSEN2 (L4/R4) sites, of puromycin-resistant single clones.
  • FIG. 10 shows the sequencing chromatogram of target regions of the HSPA family genes in pooled cells transfected with or without a donor.
  • the sgRNA-targeting sites are shaded, and target regions containing donor insertions are not included in these sequencing analyses.
  • FIGS. 11 A- 11 B show identification of single clones with inserted donor at target sites of five HSPA family genes, HSPA1A, HSPA1B, HSPA1L, HSPA6 and HSPA2.
  • FIG. 11 A PCR verification results of linear donor integration of puromycin-resistant single clones at all the five gene loci.
  • FIG. 11 B Summary of donor insertion results at four gene loci, HSPA1A, HSPA1B, HSPA1L and HSPA6.
  • FIG. 12 shows an experimental flow chart of gene knockout using a donor comprising a universal sgRNA.
  • FIG. 13 shows the efficiency verification of gene knockout using a donor comprising a universal sgRNA.
  • the present invention provides a novel donor construct and a gene knockout method.
  • the method uses a linear donor DNA to improve the efficiency of generating a gene knockout by a sequence-specific nuclease.
  • the linear donor DNA of the present invention comprises at least one target site that can be cleaved by a sequence-specific nuclease.
  • the target site comprised in the linear donor DNA is designed according to the target site in the cell genome, so that a sequence-specific nuclease that can cleave the target site in the cell genome can also cleave the target site comprised in the linear donor DNA.
  • double-strand breaks are generated at a specific target site in the cell by the sequence-specific nuclease, and at the same time the sequence-specific nuclease cleaves at least one target site contained in the linear donor DNA.
  • NHEJ non-homologous end joining
  • Subsequent selection of cells through a marker can effectively enrich cells in which a gene is knocked out by cleavage at the specific target site of the genome, thereby greatly improving the efficiency of generating the gene knockout by the sequence-specific nuclease.
  • the target site comprised in the linear donor DNA is designed according to the target site in the cell genome, and the linear donor DNA obtained is a specific linear donor.
  • the inventors further provide a universal linear donor DNA in the present invention.
  • the universal linear donor DNA comprises a universal target sequence that can be cleaved by a sequence-specific nuclease.
  • the universal target sequence is absent in the cell genome to be subjected to a gene knockout, i.e., there is no sequence, which is identical to the universal target sequence and cleavable by the sequence-specific nuclease, in the cell genome to be subjected to a gene knockout.
  • a sequence-specific nuclease and a universal linear donor DNA are introduced into a cell, a sequence-specific nuclease generates double-strand breaks (DSBs) at a specific target site in the cell, and the universal target sequence contained in the universal linear donor DNA is also cleaved by the sequence-specific nuclease through a universal gRNA that recognizes the target sequence.
  • the linear donor DNA can still be inserted into the cleaved target site in the cell genome by the non-homologous end joining (NHEJ) pathway with a higher efficiency.
  • NHEJ non-homologous end joining
  • Subsequent selection of cells through a marker can effectively enrich cells in which a gene is knocked out by cleavage at the specific target site of the genome, and can also greatly improve the efficiency of generating a gene knockout by the sequence-specific nuclease.
  • the target sequence in the universal linear donor DNA is not relevant to the gene to be knocked out, and it can be used as a universal donor for the knockout of different target genes in different cells, and can improve the efficiency of generating a gene knockout by the sequence-specific nuclease.
  • a universal linear donor DNA is particularly useful in the case of gene knockout using the Cas9/CRISPR system which targets a target sequence using a gRNA (preferably an sgRNA).
  • a gRNA preferably an sgRNA.
  • it is only necessary to construct a gRNA for a specific target site in the cell genome, without the need to specifically construct a matched linear donor DNA, i.e., a universal linear donor DNA and a gRNA targeting the universal linear donor DNA can be directly used, thereby reducing the operation complexity and improving the efficiency.
  • the mutation frequency of the target alleles is usually higher [25, 26].
  • the inventors speculate that if a donor can be inserted at a specific site in one of the target alleles, and clones that express a marker gene contained in the donor are selected, it may be possible to enrich rare events where all the alleles are modified.
  • the “gene knockout” is to realize the loss of gene functions through genome editing.
  • the gene knockout effect that is usually pursued is simultaneous knockout of two alleles, at which time the corresponding protein loses its functions and a gene knockout cell line is obtained. If only one allele is knocked out, the protein can also play its partial role, i.e., the protein functions are only down-regulated. Cells with both alleles knocked out can be enriched effectively by using the linear donor DNA and the method of the present invention.
  • the donor construct of the present invention is a double-stranded DNA.
  • the donor construct of the present invention may itself be a linear donor DNA.
  • the donor construct of the present invention may be a circular DNA molecule comprising a linear donor DNA, and when introduced into a cell, it is cleaved in the cell to produce the linear donor DNA.
  • a method for cleaving a circular donor construct in a cell to produce a linear donor DNA is well known in the art.
  • the circular construct can further comprise cleavage sites for another sequence-specific nuclease upstream of the 5′-end and downstream of the 3′-end of the linear donor DNA.
  • the method of the present invention may further comprise introducing to the cell another sequence-specific nuclease, which cleaves a sequence upstream of the 5′-end and downstream of the 3′-end of the linear donor DNA in the circular construct in the cell, thereby producing the linear donor DNA.
  • the “reverse termination codon” means the codon oriented in the opposite direction to the reading frame of the expression cassette.
  • the “forward termination codon” means the codon oriented in the same direction as the reading frame of the expression cassette.
  • the role of termination codons is that, regardless of whether the linear donor is inserted into the genome forward or backward, both the triplet termination codons can terminate endogenous and exogenous gene expression.
  • the “protective sequence” in the linear donor DNA of the present invention can be any sequence, and preferably the protective sequence is different from the target sequence in the same linear donor DNA.
  • the protective sequence can be 5-30 bp, preferably 20 bp, in length.
  • the role of the protective sequence is to protect the target sequence in the linear donor DNA from being cleaved by an enzyme (e.g., an exonuclease) in the cell.
  • the “marker gene” described herein refers to any marker gene whose expression can be selected or enriched, i.e., when the marker gene is expressed in a cell, cells expressing the marker gene can be selected and enriched in a certain manner.
  • the marker gene useful in the present invention includes, but is not limited to, a fluorescent protein gene that can be sorted by FACS after expression, or a resistance gene that can be screened by an antibiotic, or a protein gene that can be recognized by a corresponding antibody and screened by immunostaining or magnetic beads adsorption after expression.
  • the resistance gene useful in the present invention includes, but is not limited to, resistance genes against Blasticidin, Geneticin (G-418), Hygromycin B, Mycophenolic Acid, Puromycin, Zeocin or Neomycin.
  • the fluorescent protein gene useful in the present invention includes, but is not limited to, genes of Cyan Fluorescent Protein, Green Fluorescent Protein, Yellow Fluorescent Protein, Orange Fluorescent Protein, Red Fluorescent Protein, Far-Red Fluorescent Protein, or Switchable Fluorescent Proteins.
  • Examples of the sequence-specific nuclease comprises a zinc finger nuclease (ZFN).
  • the zinc finger nuclease is a non-naturally occurring and artificially modified endonuclease, which is composed of a zinc finger protein domain and a non-specific endonuclease domain.
  • the zinc finger protein domain comprises a series of Cys2-His2 zinc finger proteins in series. Each zinc finger protein recognizes and binds to a specific base triplet on the DNA strand in the 3′ to 5′ direction and a base in the 5′ to 3′ direction. Multiple zinc finger proteins can be connected in series to form a zinc finger protein group, which recognizes a stretch of specific base sequence with a strong specificity.
  • the non-specific endonuclease linked to the zinc finger protein group is derived from the DNA cleavage domain consisting of 96 amino acid residues at the carboxyl terminus of Fold.
  • Each Fokl monomer is linked to a zinc finger protein group to form a ZFN that recognizes a specific site.
  • two recognition sites are at an appropriate distance (6-8 bp)
  • two monomeric ZFNs interact with each other to produce an enzymatic digestion function, so as to achieve the purpose of site-specific DNA cleavage.
  • 8-10 zinc finger domains are designed for the target sequence.
  • sequence-specific nuclease comprises a transcription activator-like effector nuclease (TALEN).
  • the transcription activator-like effector nuclease is mainly composed of a Fok I endonuclease domain and a DNA binding domain of the TALE protein.
  • the TALE protein contains multiple peptide segment repeats, each of which comprises 33-35 amino acids, and each peptide segment recognizes one base.
  • TALENs can also cleave DNA target sequences to form DSBs, thereby activating DNA damage repair mechanisms and performing site-specific modification of the genome.
  • the Cas9/CRISPR system utilizes RNA-directed DNA binding for sequence-specific cleavage of a target DNA, in which a crRNA (CRISPR-derived RNA) binds to tracrRNA (trans-activating RNA) by base pairing to form a tracrRNA/crRNA complex, which directs the nuclease Cas9 protein to cleave the double-stranded DNA at a specific position in the target sequence that is paired with the crRNA.
  • the target sequence paired with the crRNA is usually a sequence of about 20 nucleotides located upstream of the genomic PAM (protospacer adjacent motif) site (NNG).
  • the Cas9 protein cleaves the target site by means of a guide RNA.
  • guide RNA is also known as gRNA (guide RNA).
  • a gRNA typically comprises a nucleotide on the crRNA complementary to the target sequence and an RNA scaffold formed by base pairing of the crRNA and the tracrRNA, and is capable of recognizing the target sequence paired with the crRNA.
  • the gRNA can form a complex with the Cas9 protein and guide the Cas9 protein to the target sequence for cleaving the target site therein.
  • the gRNA is commonly used in the form of an sgRNA (single guide RNA).
  • the sgRNA also known as a “single-stranded guide RNA”, is an RNA strand formed by fusing the crRNA with the trancrRNA.
  • An NgAgo nuclease can bind to a single-stranded guide DNA (gDNA) phosphorylated at the 5′-end to cleave the target sequence complementary to the gDNA, thus producing DNA double-strand breaks.
  • gDNA single-stranded guide DNA
  • the linear donor DNA of the present invention may have a target sequence only at one end, or may have target sequences at both ends, respectively.
  • the target sequences at both ends of the linear donor DNA can be different.
  • two linear donor DNAs can be provided, and each linear donor DNA comprises a corresponding target sequence, respectively; or alternatively, a linear donor DNA can be provided, each end of which comprises a corresponding target sequence.
  • an appropriate number of linear donor DNAs can be provided, and one or both ends of each linear donor DNA comprises one of the multiple different corresponding target sequences, respectively.
  • linear donor DNAs can be provided in the same number as the number of the target sites, and each linear donor DNA comprises a corresponding target sequence, respectively.
  • linear donor DNAs may be provided in an number less than the number of the target sites, wherein both ends of all or part of the linear donor DNAs comprise one of the multiple different corresponding target sequences, respectively, and each of other linear donor DNAs comprises one of the other corresponding target sequences, respectively.
  • the universal target sequence may be contained at either end or both ends.
  • the target sequence of such universal linear donor DNA is independent of the target sites to be cleaved in the cell genome, and is thus universally applicable to the case of generating a gene knockout by the cleavage of any one target site, any two target sites, or any more target sites in the cell genome.
  • the “universal target sequence” of the present invention refers to a sequence that can be cleaved by a sequence-specific nuclease.
  • the universal target sequence is absent in the cell genome to be subjected to a gene knockout, in other words, there is no sequence, which is identical to the universal target sequence and cleavable by the sequence-specific nuclease, in the cell genome to be subjected to a gene knockout.
  • the universal target sequence is different from the target sequence that is present in the cell genome and cleavable by the same sequence-specific nuclease.
  • the linear donor DNA comprising the universal target site is not specific to any target site in the cell genome, and is thus universally applicable to the gene knockout of any gene in the cell, without the need to construct a specific linear donor DNA for the gene to be knocked out and the target site in the gene.
  • the sequence-specific nuclease can be introduced into a cell in the form of a protein or its coding nucleic acid sequence (e.g., an mRNA or a cDNA).
  • a nucleic acid encoding the sequence-specific nuclease can be introduced into a cell by inclusion in a plasmid or viral vector, e.g., introduced into a cell by transfection.
  • a nucleic acid encoding the sequence-specific nuclease can also be delivered directly to a cell by electroporation, liposome, microinjection, or other means.
  • the donor construct can be delivered by any method suitable for introducing a nucleic acid into a cell, e.g., introduced into a cell by transfection.
  • an sgRNA or a gDNA should also be introduced into a cell.
  • the sgRNA or gDNA can be delivered by any method suitable for introducing an RNA or a DNA into a cell.
  • the sgRNA can be introduced into a cell in the form of an isolated RNA.
  • the isolated sgRNA can be prepared by in vitro transcription using any in vitro transcription system known in the art.
  • the sgRNA can also be introduced into a cell by a vector comprising an sgRNA coding sequence and a promoter.
  • the vector may be a viral vector or a plasmid.
  • the means for introduction into a cell can be transfection.
  • Two or more sgRNAs for different respective target sites can be introduced into a cell to direct cleavage by Cas9 at two or more different target sites in the cell genome to produce gene knockouts.
  • the two or more sgRNAs may be comprised in different vectors, or may be contained in the same vector, such as a vector comprising a pair of gRNAs (paired gRNAs), or a vector comprising more sgRNAs.
  • linear donor DNAs comprising target sequences recognized by these sgRNAs are simultaneously introduced. Since the linear donor DNA may comprise a target sequence only at the 5′-end or 3′-end, or may also comprise target sequences at both ends, respectively, the number of sgRNAs and the number of linear donor DNAs can be different, i.e., it is possible that one sgRNA corresponds to one linear donor DNA, or two sgRNAs correspond to two linear donor DNAs.
  • an sgRNA for a specific target sequence in the cell genome and an sgRNA for a universal target sequence on the universal linear donor DNA are also introduced into the cell, so as to direct the Cas9 to cleave the specific target sequence in the cell genome and the universal target sequence on the universal linear donor DNA.
  • An sgRNA for a specific target sequence in the cell genome and an sgRNA for a universal target sequence on the universal linear donor DNA may be conprised in different vectors, or may be comprised in the same vector.
  • An sgRNA for a specific target sequence in the cell genome may be one sgRNA or more sgRNAs, such as two, three, or more gRNAs.
  • the more than one sgRNA may target different specific target sites in the cell genome respectively, so as to achieve simultaneous cleavage on different target sites in the cell genome.
  • the knockout of multiple genes such as of two, three or more genes, can be achieved.
  • a plurality of sgRNAs for a plurality of respective specific target sites in the cell genome and an sgRNA for a universal target sequence on the universal linear donor DNA may be introduced into the cell, so as to direct the Cas9 to cleave the plurality of specific target sites in the cell genome and the universal target sequence on the universal linear donor DNA.
  • the plurality of specific target sequences are located on different genes respectively, thereby achieving the multi-gene knockout.
  • the plurality of sgRNAs for the plurality of respective specific target sites in the cell genome may be comprised in different vectors, or may be comprised in the same vector.
  • any one or more sgRNAs of the plurality of sgRNAs for the plurality of respective specific target sites in the cell genome, and an sgRNA for a universal target sequence on the universal linear donor DNA may be comprised in different vectors, or may be comprised in the same vector.
  • the universal target sequence on the universal donor construct DNA is preferably 5′-GTACGGGGCGATCATCCACA-3′ (SEQ ID NO:1) or 5′-AATCGACTCGAACTTCGTGT-3′ (SEQ ID NO:2).
  • a Cas9, an sgRNA and a linear donor DNA can be introduced into a cell simultaneously; or for instance, a Cas9 can be first introduced into a cell, and then an sgRNA and a linear donor DNA are introduced into the cell.
  • the cell is co-transfected with a Cas9-containing vector, an sgRNA-containing vector, and a linear donor DNA.
  • a Cas9 and an sgRNA are assembled in vitro into a protein-RNA complex, which is used to co-transfect the cell with a linear donor DNA.
  • a Cas9 and an sgRNA are stably expressed in the cell by lentivirus, and the cell is transfected with a linear donor DNA.
  • a Cas9 is first stably expressed in the cell, and the cell is then co-transfected with an sgRNA-containing vector and a linear donor DNA.
  • a sequence-specific nuclease may be in the form of a protein or its coding nucleic acid sequence (e.g., an mRNA or a cDNA), such as in the form of a plasmid or viral vector comprising a nucleic acid encoding the sequence-specific nuclease.
  • an sgRNA may be in the form of an isolated RNA, or in the form of a vector comprising an sgRNA coding sequence and a promoter, such as a viral vector or a plasmid vector.
  • the cell described herein can be any eukaryotic cell, such as an isolated animal cell, e.g., a totipotent cell, a pluripotent cell, an adult stem cell, a fertilized egg, a somatic cell, or the like.
  • the cell is a vertebrate cell.
  • the cell is a mammalian cell.
  • the cell is a human cell.
  • the cell is a cell from a cow, a goat, a sheep, a cat, a dog, a horse, a rodent, fish, and a primate.
  • the rodent comprises mice, rats, and rabbits.
  • the method of the present invention can be used to perform targeted gene knockout in a single gene or multiple genes in a cell, such as two, three, four, five or more targeted gene knockouts.
  • Targeted gene knockout for multiple genes can be performed simultaneously or successively.
  • sequence-specific nucleases or sequence-specific nuclease systems for two or more target genes can be introduced into a cell and then cells are subjected to enrichment screening.
  • a sequence-specific nuclease(s) or a sequence-specific nuclease system(s) for one or more target genes can be first introduced into a cell and cells are subjected to enrichment screening, and then a sequence-specific nuclease(s) or a sequence-specific nuclease system(s) for other target genes can be introduced into the cell and the cells are subjected to enrichment screening.
  • Different marker/marker genes can be used for different target genes.
  • two or more sgRNAs for different respective target sites can be introduced into a cell, and a linear donor DNA(s) comprising target sequences recognized by these sgRNAs is(are) simultaneously introduced, as previously mentioned.
  • a linear donor DNA(s) comprising target sequences recognized by these sgRNAs is(are) simultaneously introduced, as previously mentioned.
  • target sites are located in different genes, knockout of multiple genes can be achieved.
  • Target sequences in an sgRNA and a linear donor DNA can also be designed by using the consensus sequence of two or more genes in the cell genome.
  • an sgRNA recognizing a single specific target site in the cell genome can be introduced into a cell, and a linear donor DNA comprising a target sequence recognized by the sgRNA is simultaneously introduced, wherein the target sequence recognized by the sgRNA is the consensus sequence of two or more genes in the cell genome, in the condition that the consensus sequence has no more than one base difference from the sequences in any of the two or more genes at positions corresponding to the consensus sequence.
  • a two-base difference may disrupt recognition by an sgRNA, as demonstrated in Example 7.
  • the target gene edited with the linear donor DNA of the present invention targets is not particularly limited, as long as double-strand breaks can be produced on it by the Cas9/CRISPR system.
  • the target gene may be an exon, an intron or a regulatory sequence, or any combination thereof.
  • sgRNAs targeting the first exon of the ANTXR1 gene in HeLa cells were designed, and their efficiency in producing deletions or insertion mutations (Indels) at target sites was verified by T7E1 assay. The verification results are shown in Table 1.
  • the target sequence that sgRNA1 ANTXR1 targets is referred to as sg1 in this example, and the target sequence that sgRNA2 ANTXR1 targets is referred to as sg2 in this example.
  • DonorANTXR1-sg2 and DonorANTXR1-pg were constructed, the structures of which are shown in FIG. 1 A .
  • Donor ANTXR1-sg2 comprises, from 5′-end to 3′- end: a 20-bp protective sequence, sg2, a reverse termination codon, CMV promoter-driven puromycin-resistant gene, a forward termination codon, and a 20-bp protective sequence, respectively.
  • Donor ANTXR1-pg comprises, from 5′-end to 3′-end: a 20-bp protective sequence, sg 1, a reverse termination codon, CMV promoter-driven puromycin-resistant gene, a forward termination codon, sg2, and a 20-bp protective sequence, respectively.
  • a linear donor DNA (Donor no cut ) as a control comprises, from 5′-end to 3′-end: a 20-bp protective sequence, a 20-bp random sequence, a reverse termination codon, CMV promoter-driven puromycin-resistant gene, a forward termination codon, and a 20-bp protective sequence, respectively.
  • the random sequence is different from sg1 or sg2.
  • HeLa cells were co-transfected with a Cas9-expressing plasmid, sgRNA2 ANTXR1 or pgRNA ANTXR1 , and the corresponding donors.
  • a Cas9-expressing plasmid sgRNA2 ANTXR1 or pgRNA ANTXR1
  • HeLa cells were transfected with linear donor DNAs (Donor ANTXR1-sg2 , Donor ANTXR1-pg , and Donor no cut ) alone.
  • To the cells puromycin was added for resistance screening.
  • a pooled population and single clones were obtained and stained with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltertazolium bromide). The results are shown in FIG. 1 B .
  • puromycin-resistant (puro+) cell clones were obtained from samples receiving sgRNA2 ANTXR1 and its corresponding donor Donor ANTXR1-sg2 , and from samples receiving pgRNA ANTXR1 and its corresponding donor Donor ANTXR1-pg . Only a few puromycin-resistant clones were produced by transfection with donors alone, probably because integration of linear donors into the chromosome was rare and random. In addition, co-transfection using the control donor Donor no cut with the Cas9-expressing plasmid and sgRNA2 ANTXR1 also failed to produce a significant number of puro+clones (see the rightmost panel in FIG.
  • HeLa cells were co-transfected using a Cas9-expressing plasmid and sgRNA2 ANTXR1 or pgRNA ANTXR1 , with or without the corresponding donors.
  • a pooled population and single clones were obtained by screening with puromycin (1 ⁇ g/ml).
  • the plasmid expressing the puromycin-resistant gene is used for co-transfection instead, when the corresponding donors are not added.
  • ANTXR1 gene knockout in HeLa cells results in resistance of the cells to chimeric anthrax toxin (PA/LFnDTA) [17]
  • PA/LFnDTA chimeric anthrax toxin
  • PA/LFnDTA PA: 150 ng/ml
  • LFnDTA 100 ng/ml
  • FIG. 2 A Images of different cells after being treated with PA/LFnDTA are shown in FIG. 2 A .
  • the ANTXR1 knockout efficiency was determined by calculating the percentage of cells with the toxin resistance in the puro+pooled population, as shown in FIG. 1 C .
  • a donor with a single- or double-cleavage site is capable of greatly improving the selection of cells with a modification at a target site, for convenience, in this example, only a single-cleavage donor was adopted.
  • sgRNAs targeting the HBEGF gene in HeLa cells were designed, and their efficiency in producing Indels at target sites was verified by T7E1 assay. The verification results are shown in Table 3.
  • the target sequence that sgRNA1 HBEGY targets is referred to as sg 1 in this example, and the target sequence that sgRNA2 HBEGF targets is referred to as sg2 in this example.
  • a linear donor DNA (Donor HBEGF-sg1 ) was constructed, the structure of which is shown in FIG. 3 A .
  • Donor HBEGY-sg1 comprises, from 5′-end to 3′- end: a 20-bp protective sequence, sg 1, a reverse termination codon, CMV promoter-driven puromycin-resistant gene, a forward termination codon, and a 20-bp protective sequence, respectively.
  • HeLa cells were co-transfected with a Cas9-expressing plasmid, sgRNA1 HBEGF , and its corresponding donor Donor HBEGF-sg1 .
  • sgRNA1 HBEGF a Cas9-expressing plasmid
  • donor Donor HBEGY-sg1 a Cas9-expressing plasmid
  • Puromycin was added to the cells for resistance screening.
  • a pooled population and single clones were obtained and stained with MTT. The results are shown in FIG. 3 B .
  • HeLa cells were co-transfected using a Cas9-expressing plasmid and sgRNA1 HBEGF , with or without its corresponding donor Donor HBEGF-sg1 .
  • a pooled population and single clones were obtained by screening with puromycin (1 ⁇ g/ml).
  • the plasmid expressing the puromycin-resistant gene is used for co-transfection insdead, when the corresponding donor is not added.
  • HBEGF gene encodes a diphtheria toxin (DT) receptor
  • DT diphtheria toxin
  • FIG. 3 C Images of different cells after being treated with DT are shown in FIG. 3 C .
  • the HBEGF knockout efficiency was determined by calculating the percentage of cells with DT resistance in the puro+pooled population, as shown in FIG. 3 D .
  • the use of the linear donor DNA greatly improves the HBEGF gene knockout efficiency compared with the use of sgRNA1 HBEGF alone.
  • sgRNA2 HBEGF targeting the HBEGF gene in HEK293T cells was designed and a linear donor DNA (Donor HBEGF-sg2 ) was constructed.
  • the donor comprises, from 5′-end to 3′-end: a 20-bp protective sequence, sg2, a reverse termination codon, CMV promoter-driven EGFP gene, a forward termination codon, and a 20-bp protective sequence, respectively, as shown in FIG. 4 A .
  • HEK293T cells were co-transfected using a Cas9-expressing plasmid and sgRNA2 HBEGF , with or without its corresponding donor Donor HBEGF-sg2 .
  • Cells were screened by FACS. The group added the donor was screened for EGFP-positive cells by FACS, while the group didn't add the donor was screened for mCherry-positive cells by FACS.
  • FACS-selected cells were treated with DT (40 ng/ml) to compare the effects of the linear donor DNAs on the HBEGF knockout efficiency. Images of different cells after being treated with DT are shown in FIG. 4 B .
  • the HBEGF knockout efficiency was determined by calculating the percentage of cells with DT resistance in the EGFP-positive cells, as shown in FIG. 4 C .
  • the use of the linear donor DNA greatly improves the HBEGF gene knockout efficiency compared with the use of sgRNA2 HBEGF alone.
  • the HeLa oc cell line stably expressing Cas9 was established according to the existing method [17].
  • sgRNA1 ANTXR1 and sgRNA2 ANTXR1 Two sgRNAs (sgRNA1 ANTXR1 and sgRNA2 ANTXR1 ) targeting the ANTXR1 gene in HeLa oc cells were designed, and three linear donor DNAs (Donor ANTXR1-sg1 , Donor ANTXR1-sg2 and Donor ANTXR1-pg ) were constructed, as shown in FIG. 5 A .
  • the target sequence that sgRNA1 ANTXR1 targets is referred to as sg 1 in this example, and the target sequence that sgRNA2 ANTXR1 targets is referred to as sg2 in this example.
  • Donor ANTXR1-sg1 comprises, from 5′-end to 3′- end: a 20-bp protective sequence, sg 1, a reverse termination codon, CMV promoter-driven puromycin-resistant gene, a forward termination codon, and a 20-bp protective sequence, respectively.
  • Donor ANTXR1-sg2 comprises, from 5′-end to 3′-end: a 20-bp protective sequence, sg2, a reverse termination codon, CMV promoter-driven puromycin-resistant gene, a forward termination codon, and a 20-bp protective sequence, respectively.
  • Donor ANTXR1-pg comprises, from 5′-end to 3′-end: a 20-bp protective sequence, sg 1, a reverse termination codon, CMV promoter-driven puromycin-resistant gene, a forward termination codon, sg2, and a 20-bp protective sequence, respectively.
  • HeLa oc cells were co-transfected with a Cas9-expressing plasmid, sgRNA1 ANTXR1 or sgRNA2 ANTXR1 or pgRNA ANTXR1 , and the corresponding donors.
  • a Cas9-expressing plasmid sgRNA1 ANTXR1 or sgRNA2 ANTXR1 or pgRNA ANTXR1 , and the corresponding donors.
  • HeLa oc cells were transfected with linear donor DNAs (Donor ANTXR1-SG1 , Donor ANTXR1-sg2 , and Donor ANTXR1-pg ) alone.
  • HeLa oc cells were co-transfected using a Cas9-expressing plasmid and sgRNA1 ANTXR1 or sgRNA2 ANTXR1 or pgRNA ANTXR1 , with or without the corresponding donors, and screened with puromycin (1 ⁇ g/ml).
  • the cells obtained by screening were treated with PA/LFnDTA. Images of different cells treated with PA/LFnDTA are shown in FIG. 5 C .
  • the ANTXR1 knockout efficiency was determined by calculating the percentage of cells with the toxin resistance in the puro+pooled population, as shown in FIG. 5 D .
  • the use of linear donor DNAs greatly improves the ANTXR1 gene knockout efficiency compared to the use of sgRNAs alone.
  • PCR verification was performed on the integration site of donor Donor ANTXR1-sg1 in the ANTXR1 gene. It was found that most of the puro+clones contain donor inserts at the sgRNA-targeting sites ( FIG. 5 E and FIG. 5 F ), and most of the cells carrying donor fragments are real gene knockout clones ( FIG. 5 F ).
  • a PCR fragment of about 500 bp (the length corresponding to the wild type ANTXR1 gene) and a PCR fragment of about 1.8 kb (the length corresponding to the wild type ANTXR1 gene plus a donor insert) were subjected to genome sequencing. The results are shown in Table 4.
  • the off-target insertions in single clones and pooled clones were verified by splinkerette PCR analysis after puromycin selection. If a correct donor insertion in the ANTXR1 gene is present, amplification with primers Splink2/R1 and Splink2/R2 will result in 711- and 927-bp products, respectively (see FIG. 6 A ).
  • sgRNA1 HBEGF and sgRNA2 HBEGF targeting the HBEGF gene in HeLa oc cells were designed, and three linear donor DNAs (Donor HBEGF-sg1 , Donor HBEGF-sg2 and Donor HBEGF-pg ) were constructed, as shown in FIG. 7 A .
  • the target sequence that sgRNA1 HBEGF targets is referred to as sg 1 in this example, and the target sequence that sgRNA2 HBEGF targets is referred to as sg2 in this example.
  • Donor HBEGF-sg1 comprises, from 5′-end to 3′- end: a 20-bp protective sequence, sg 1, a reverse termination codon, CMV promoter-driven puromycin-resistant gene, a forward termination codon, and a 20-bp protective sequence, respectively.
  • Donor HBEGF-sg2 comprises, from 5′-end to 3′-end: a 20-bp protective sequence, sg2, a reverse termination codon, CMV promoter-driven puromycin-resistant gene, a forward termination codon, and a 20-bp protective sequence, respectively.
  • Donor HBEGF-pg comprises, from 5′-end to 3′-end: a 20-bp protective sequence, sg 1, a reverse termination codon, CMV promoter-driven puromycin-resistant gene, a forward termination codon, sg2, and a 20-bp protective sequence, respectively.
  • HeLa oc cells were co-transfected with a Cas9-expressing plasmid, sgRNA1 HBEGF or sgRNA2 HBEGF or pgRNA HBEGF , and the corresponding donors.
  • a Cas9-expressing plasmid sgRNA1 HBEGF or sgRNA2 HBEGF or pgRNA HBEGF
  • HeLa oc cells were transfected with linear donor DNAs (Donor HBEGF-sg1 , Donor HBEGF-sg2 and Donor HBEGF-pg ) alone. Puromycin was added to the cells for resistance screening. The results are shown in FIG. 7 B .
  • PCR verification was performed on the integration site of donor Donor HBEGF-sg1 in the HBEGF gene.
  • the L2/R2 primer sequences used in PCR amplification are shown in Table 5. It was found that most of the puro+clones contain donor inserts at the sgRNA-targeting sites ( FIG. 7 C and FIG. 7 D ), and most of the cells carrying donor fragments are real gene knockout clones ( FIG. 7 D ).
  • sgRNAs targeting these two target genes respectively were designed, and their efficiency in producing indels at target sites was verified by T7E1 assay. The results are shown in Table 6.
  • the target sequence that sgRNA PSEN1 targets is referred to as sg PSEN1 in this example, and the target sequence that sgRNA PSEN2 targets is referred to as sg PSEN2 in this example.
  • Donor PSEN1 +Donor PSEN2 Two types of donors were constructed. One type had two separate donors (Donor PSEN1 +Donor PSEN2 ), and each donor had a corresponding sgRNA target sequence; and the other type of donor (Donor PSEN ) had two sgRNA-targeting sequences at both ends, respectively, as shown in FIG. 8 A .
  • Donor PSEN1 or Donor PSEN2 had a cleavage site for sgRNA PSEN1 or sgRNA PsEN2 at the 5′-end.
  • Donor PSEN had a cleavage site for sgRNA PSEN1 at the 5′-end and a cleavage site for sgRNA PSEN2 at the 3′-end.
  • the donors include
  • Donor PSEN 1 comprises, from 5′-end to 3′-end: a 20-bp protective sequence, sg PSEN1 , a reverse termination codon, CMV promoter-driven puromycin-resistant gene, a forward termination codon, and a 20-bp protective sequence, respectively.
  • Donor PSEN 2 comprises, from 5′-end to 3′-end: a 20-bp protective sequence, sg PSEN2 , a reverse termination codon, CMV promoter-driven puromycin-resistant gene, a forward termination codon, and a 20-bp protective sequence, respectively.
  • Donor PSEN comprises, from 5′-end to 3′-end: a 20-bp protective sequence, sg PSEN1 , a reverse termination codon, CMV promoter-driven puromycin-resistant gene, a forward termination codon, Sg PSEN2 , and a 20-bp protective sequence, respectively.
  • HeLa oc cells were co-transfected with a Cas9-expressing plasmid, sgRNA PSEN1 or sgRNA PSEN2 ; or HeLa oc cells were co-transfected with a Cas9-expressing plasmid and pgRNA PSEN to produce indels at specific sites of the PSEN1 and PSEN2 genes.
  • T7E1 analysis was performed on the indels production efficiency [26] (see Table 7 for the primers used). The results are shown in FIG. 9 A , and their co-transfections all show only ordinary activity.
  • HeLa oc cells were co-transfected with a Cas9-expressing plasmid, pgRNA PSEN , and Donor PSEN ; or HeLa oc cells were co-transfected with a Cas9-expressing plasmid, pgRNA PSEN , and Donor PSEN1 +Donor PSEN2 .
  • puromycin was added for resistance screening to obtain puro+clones (see FIG. 9 B ). Similar to the results of the previous examples, the donors plus pgRNAs obtained a large number of puro+clones.
  • the HSPA gene family in HeLa oc cells was selected, which includes five homologous genes, HSAPA1A, HSPA1B, HSBA1L, HSPA6 and HSPA2.
  • the sgRNA HSPA simultaneously targeting HSAPA1A,HSPA1B and HSBA1L was designed.
  • the target sequence that the sgRNA targets has a mismatch with the corresponding sequence in HSPA6 and two mismatches with HSPA2. These are shown in FIG. 8 D .
  • a linear donor Donor HSPA was constructed, comprising, from 5′-end to 3′-end: a 20-bp protective sequence, sg HSPA , a reverse termination codon, CMV promoter-driven puromycin-resistant gene, a forward termination codon, and a 20-bp protective sequence, respectively ( FIG. 8 D ).
  • HeLa oc cells were co-transfected using a Cas9-expressing plasmid and sgRNA HSPA , with or without its corresponding donor Donor HSPA , indels were triggered, and resistance screening was performed by puromycin.
  • the group without the donor added was co-transfected with a plasmid expressing a puromycin-resistant gene instead.
  • the indels efficiency for all five genes was evaluated by T7E1 assay (see Table 8 for the primers used). The results are shown in FIG. 8 E .
  • the use of donor HSPA increased the mutation rate at the HSPA1A site by approximately 5.5 times, increased the mutation rate at the HSPA1B site by approximately 6.1 times, increased the mutation rate at the HSPA1L site by approximately 3.4 times, and increased the mutation rate at the HSPA6 site by approximately 6.6 times.
  • no indels was detected in the HSPA2 gene, regardless of whether the donor was used, indicating that the two mismatches completely disrupt the recognition by sgRNA HSPA .
  • selection using a donor did not increase the risk of off-target effects.
  • the target regions of HSPA family genes in pooled population co-transfected with and without the donor were sequenced. The results are shown in FIG. 10 . The results showed that the cell pool sequencing results are consistent with the results of T7E1 assay, regardless of whether there is donor transfection at the HSPA family gene loci.
  • the T7E1 assay demonstrates that the selected pooled clones are highly rich in cells carrying target mutations, and the enrichment factor is approximately 753 (5.5*6.1*3.4*6.6) compared with traditional methods without using a donor. Considering that this calculation does not consider genes with donor insertions, the actual efficiency is even higher.
  • Predicted gene sgRNA Target Sequence (5′ to 3′) knockout efficiency sgRNA Universal _1 GTACGGGGCGATCATCCACACGG 0.982784325 (SEQ ID NO: 25) sgRNA Universal _2 GCAAAAGTGGCATAAAACCGCGG 0.971302462 (SEQ ID NO: 26) sgRNA Universal _3 TATCGCTTCCGATTAGTCCGCGG 0.96832667 (SEQ ID NO: 27) sgRNA Universal _4 CTATCTCGAGTGGTAATGCGCGG 0.966411034 (SEQ ID NO: 28) sgRNA Universal _5 GTAGCTGCTGTAAATCGCATCGG 0.963330804 (SEQ ID NO: 29) sgRNA Universal _6 TATACCAGACCACAGCGCCGCGG 0.963267571 (SEQ ID NO: 30) sgRNA Universal _7 GCACGAGGTGAACAGCCGCTCGG 0.960224565 (SEQ ID NO: 31) sgRNA
  • linear donor DNAs comprise, from 5′-end to 3′-end: a 20-bp protective sequence, a target sequence that sgRNA Universal_1 ⁇ 10 targets, a reverse termination codon, CMV promoter-driven puromycin-resistant gene, a forward termination codon, and a 20-bp protective sequence, respectively.
  • sgRNA CSPG4 targets CSPG4, the receptor of TcdB toxin, while sgRNA Universal_1 ⁇ 10 targets the target sequence in the corresponding donor DNA
  • the cell line used in the transfection experiment was a cell line stably expressing Cas9 (SC-8).
  • SC-8 cells were co-transfected with ten tandem plasmids Plasmid pgRNA_Universal_1 ⁇ 10 and the corresponding donor DNAs (Donors sgRNA_Universal_1 ⁇ 10puro ).
  • SC-8 cells were co-transfected with ten linear donor DNAs (Donors sgRNA_Universal_1 ⁇ 10puro ) alone. Puromycin was added to the cells for resistance screening. A pooled population was obtained. The screening results are shown in the following table.
  • TcdB toxin was added to the four pooled clones for screening, and the cell survival was observed after 23 hours.
  • the experimental results are shown in FIG. 13 . It can be seen that 23 hours after the addition of TcdB toxin, the cell survival rates corresponding to the experimental groups with sgRNA Universal_1 and sgRNA Universal_9 added were significantly higher than those of the other two groups, indicating that sgRNA Universal_1 and sgRNA Universal_9 achieve higher gene knockout efficiency.
  • HeLa, HeLa oc and HEK293T cells were maintained in a Dulbecco's modified Eagle's medium (DMEM, 10-013-CV, Corning, Tewksbury, MA, USA) supplemented with 10% fetal bovine serum (FBS, Lanzhou Bailing Biotechnology Co., Ltd., Lanzhou, China) at a temperature of 37° C., and supplied with 5% CO2.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • X-tremeGENE HP 06366546001, Roche, Mannheim, Germany
  • Opti-MEM I Reduced Serum Medium 31985088, Thermo Fisher Scientific, Grand Island, N.Y., USA. The mixture was incubated for 15 minutes at room temperature and then added to the cells.
  • the oligonucleotide of each sgRNA coding sequence was designed separately (see Table 9) and synthesized (Beijing Ruibo Xingke Biotechnology Co., Ltd.).
  • Oligonucleotides were dissolved to a concentration of 10 ⁇ M in 1 ⁇ TE, and the paired oligonucleotides were mixed with TransTaq HiFi Buffer II (K10222, Beijing TransGen Biotech Co., Ltd.), heated to 95° C. for 3 minutes, and then slowly cooled to 4° C. These annealed oligonucleotide pairs were phosphorylated for 30 minutes at 37° C. After heat inactivation, the product was ligated into the sgRNA backbone vector using the “Golden Gate” method.
  • the scaffold sequence of the gRNA and the U6 promoter were amplified with primers comprising two gRNA coding sequences (Table 5), and the PCR product was then purified and ligated into the sgRNA backbone vector using the “Golden Gate” method.
  • the sgRNA backbone vector of the present invention has modifications in the sgRNA backbone [38], and has the EGFP sequence replaced with the mCherry coding sequence.
  • Genomic DNAs were extracted using a DNeasy Blood & Tissue kit (69504, Qiagen, Hilden, Germany), and the genomic region comprising the gRNA target sequence was subjected to PCR amplification.
  • the primer sequences used in the assay are shown in Table 2, Table 5, Table 7, and Table 8. 300-500 ng of PCR product obtained using these primer sequences was mixed with 10 ⁇ NEB Buffer2 in a 50 ⁇ l of system, heated at 95° C. for 3 minutes, and slowly cooled to room temperature. The resulting product was incubated with 0.5 ⁇ l of T7E1 for 15 min at 37° C. for agarose gel electrophoresis. The electropherogram was analyzed by Image J image analysis software for the band cleavage efficiency which indicates the efficiency of generating Indels by sgRNAs.
  • a donor sequence comprising a CMV-driven puromycin-resistant gene or EGFP gene, and termination codon sequences were pre-produced, and cloned into the pEASY-T5-Zero clone vector (CT501-02, Beijing TransGen Biotech Co., Ltd.) as a universal template.
  • the template was amplified using primers comprising sgRNA cleavage target sites and protective sequences. The primer sequences are shown in Table 10.
  • HeLa oc cells were transfected with 1 ⁇ g of purified linear donor PCR product and 1 ⁇ g of sgRNA/pgRNA, and treated with 1 ⁇ g/ml puromycin two weeks after transfection.
  • HeLa and HEK293T cells were transfected with 1 ⁇ g of a donor, 0.5 ⁇ g of sgRNA/pgRNA and 0.5 ⁇ g of Cas9 plasmid. The cells were then treated with 1 ⁇ g/ml puromycin two weeks after transfection, or determined to be EGFP positive by the fluorescence activated cell sorting (FACS), depending on which type of donor was used.
  • FACS fluorescence activated cell sorting
  • the splinkerette PCR method has been previously reported (Potter, C. J. & Luo, L. Splinkerette PCR for mapping transposable elements in Drosophila . PLoS One 5, e10168 (2010); Uren, A. G. et al. A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites. Nat Protoc 4, 789-798 (2009); and Yin, B. & Largaespada, D.A. PCR-based procedures to isolate insertion sites of DNA elements. Biotechniques 43, 79-84 (2007)). The primer and adaptor sequences used are shown in Table 11.

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Citations (34)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011146121A1 (en) 2010-05-17 2011-11-24 Sangamo Biosciences, Inc. Novel dna-binding proteins and uses thereof
WO2013102290A1 (zh) 2012-01-04 2013-07-11 清华大学 特异识别含有5-甲基化胞嘧啶的dna的方法
WO2013176772A1 (en) 2012-05-25 2013-11-28 The Regents Of The University Of California Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription
WO2014018423A2 (en) 2012-07-25 2014-01-30 The Broad Institute, Inc. Inducible dna binding proteins and genome perturbation tools and applications thereof
CN103668472A (zh) 2013-12-31 2014-03-26 北京大学 利用CRISPR/Cas9系统构建真核基因敲除文库的方法
US8697359B1 (en) 2012-12-12 2014-04-15 The Broad Institute, Inc. CRISPR-Cas systems and methods for altering expression of gene products
WO2014065596A1 (en) 2012-10-23 2014-05-01 Toolgen Incorporated Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof
WO2014130955A1 (en) 2013-02-25 2014-08-28 Sangamo Biosciences, Inc. Methods and compositions for enhancing nuclease-mediated gene disruption
US20140273226A1 (en) 2013-03-15 2014-09-18 System Biosciences, Llc Crispr/cas systems for genomic modification and gene modulation
US20140273233A1 (en) 2013-03-15 2014-09-18 Sigma-Aldrich Co., Llc Crispr-based genome modification and regulation
WO2014144592A2 (en) 2013-03-15 2014-09-18 The General Hospital Corporation Using truncated guide rnas (tru-grnas) to increase specificity for rna-guided genome editing
WO2014204724A1 (en) 2013-06-17 2014-12-24 The Broad Institute Inc. Delivery, engineering and optimization of tandem guide systems, methods and compositions for sequence manipulation
WO2014206568A1 (en) 2013-06-26 2014-12-31 Universität Konstanz Direct, programmable detection of epigenetic dna cytosine modifications using tal effectors
US8932814B2 (en) 2012-12-12 2015-01-13 The Broad Institute Inc. CRISPR-Cas nickase systems, methods and compositions for sequence manipulation in eukaryotes
US20150037809A1 (en) 2012-03-23 2015-02-05 Cellectis, S.A. Method to overcome dna chemical modifications sensitivity of engineered tale dna binding domains
CN104651399A (zh) 2014-12-31 2015-05-27 广西大学 一种利用CRISPR/Cas系统在猪胚胎细胞中实现基因敲除的方法
WO2016011080A2 (en) 2014-07-14 2016-01-21 The Regents Of The University Of California Crispr/cas transcriptional modulation
CN105316341A (zh) 2015-12-08 2016-02-10 浙江理工大学 一种LncRNA及其在作为前列腺癌检测标记物或前列腺癌预后复发标记物中的应用
WO2016142719A1 (en) 2015-03-12 2016-09-15 Genome Research Limited Biallelic genetic modification
KR20160118987A (ko) 2015-04-01 2016-10-12 한양대학교 산학협력단 LincRNA 삭제를 위한 sgRNA 쌍
WO2016182893A1 (en) 2015-05-08 2016-11-17 Teh Broad Institute Inc. Functional genomics using crispr-cas systems for saturating mutagenesis of non-coding elements, compositions, methods, libraries and applications thereof
WO2016184989A1 (en) 2015-05-19 2016-11-24 Kws Saat Se Methods for the in planta transformation of plants and manufacturing processes and products based and obtainable therefrom
CN106232823A (zh) 2014-02-18 2016-12-14 杜克大学 灭活病毒复制的组合物及其制备和使用方法
WO2016205745A2 (en) 2015-06-18 2016-12-22 The Broad Institute Inc. Cell sorting
CN106637421A (zh) 2016-10-28 2017-05-10 北京大学 双sgRNA文库的构建及其应用于高通量功能性筛选研究的方法
US20170198302A1 (en) * 2015-11-17 2017-07-13 The Chinese University Of Hong Kong Methods and systems for targeted gene manipulation
CN107090466A (zh) 2017-04-20 2017-08-25 清华大学 双sgRNA表达质粒及其文库的构建方法
WO2017214460A1 (en) 2016-06-08 2017-12-14 Agilent Technologies, Inc. High specificity genome editing using chemically modified guide rnas
CN107513538A (zh) 2016-06-17 2017-12-26 北京大学 基因敲除方法
WO2018154027A1 (en) 2017-02-22 2018-08-30 Cancer Research Technology Ltd. Cell labelling, tracking and retrieval
WO2019024081A1 (zh) 2017-08-04 2019-02-07 北京大学 特异性识别甲基化修饰dna碱基的tale rvd及其应用
WO2019191876A1 (en) 2018-04-02 2019-10-10 Peking University Method for Screening and Identifying Functional lncRNAs
WO2020125762A1 (en) 2018-12-20 2020-06-25 Peking University Compositions and methods for highly efficient genetic screening using barcoded guide rna constructs
WO2020192712A1 (en) 2019-03-26 2020-10-01 Peking University Method for identifying functional elements

Patent Citations (49)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011146121A1 (en) 2010-05-17 2011-11-24 Sangamo Biosciences, Inc. Novel dna-binding proteins and uses thereof
JP2013529083A (ja) 2010-05-17 2013-07-18 サンガモ バイオサイエンシーズ， インコーポレイテッド 新規のｄｎａ結合タンパク質及びその使用
CN103025344A (zh) 2010-05-17 2013-04-03 桑格摩生物科学股份有限公司 新型dna-结合蛋白及其用途
US20140134740A1 (en) 2010-05-17 2014-05-15 Sangamo Biosciences, Inc. Novel dna-binding proteins and uses thereof
WO2013102290A1 (zh) 2012-01-04 2013-07-11 清华大学 特异识别含有5-甲基化胞嘧啶的dna的方法
US20150037809A1 (en) 2012-03-23 2015-02-05 Cellectis, S.A. Method to overcome dna chemical modifications sensitivity of engineered tale dna binding domains
JP2015510772A (ja) 2012-03-23 2015-04-13 セレクティスＣｅｌｌｅｃｔｉｓ 工学的に操作されたｔａｌｅｄｎａ結合性ドメインの化学的ｄｎａ改変感受性を変更する方法
US10113167B2 (en) 2012-05-25 2018-10-30 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
WO2013176772A1 (en) 2012-05-25 2013-11-28 The Regents Of The University Of California Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription
WO2014018423A2 (en) 2012-07-25 2014-01-30 The Broad Institute, Inc. Inducible dna binding proteins and genome perturbation tools and applications thereof
WO2014065596A1 (en) 2012-10-23 2014-05-01 Toolgen Incorporated Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof
US8697359B1 (en) 2012-12-12 2014-04-15 The Broad Institute, Inc. CRISPR-Cas systems and methods for altering expression of gene products
US8932814B2 (en) 2012-12-12 2015-01-13 The Broad Institute Inc. CRISPR-Cas nickase systems, methods and compositions for sequence manipulation in eukaryotes
WO2014130955A1 (en) 2013-02-25 2014-08-28 Sangamo Biosciences, Inc. Methods and compositions for enhancing nuclease-mediated gene disruption
US20140273233A1 (en) 2013-03-15 2014-09-18 Sigma-Aldrich Co., Llc Crispr-based genome modification and regulation
WO2014144761A2 (en) 2013-03-15 2014-09-18 The General Hospital Corporation Increasing specificity for rna-guided genome editing
WO2014144592A2 (en) 2013-03-15 2014-09-18 The General Hospital Corporation Using truncated guide rnas (tru-grnas) to increase specificity for rna-guided genome editing
US20140273226A1 (en) 2013-03-15 2014-09-18 System Biosciences, Llc Crispr/cas systems for genomic modification and gene modulation
WO2014204724A1 (en) 2013-06-17 2014-12-24 The Broad Institute Inc. Delivery, engineering and optimization of tandem guide systems, methods and compositions for sequence manipulation
CN106062197A (zh) 2013-06-17 2016-10-26 布罗德研究所有限公司 用于序列操纵的串联指导系统、方法和组合物的递送、工程化和优化
WO2014206568A1 (en) 2013-06-26 2014-12-31 Universität Konstanz Direct, programmable detection of epigenetic dna cytosine modifications using tal effectors
US20160138094A1 (en) 2013-06-26 2016-05-19 Universität Konstanz Direct, Programmable Detection of Epigenetic DNA Cytosine Modifications using TAL Effectors
CN103668472A (zh) 2013-12-31 2014-03-26 北京大学 利用CRISPR/Cas9系统构建真核基因敲除文库的方法
CN106232823A (zh) 2014-02-18 2016-12-14 杜克大学 灭活病毒复制的组合物及其制备和使用方法
US20170049909A1 (en) 2014-02-18 2017-02-23 Duke University Compositions for the inactivation of virus replication and methods of making and using the same
WO2016011080A2 (en) 2014-07-14 2016-01-21 The Regents Of The University Of California Crispr/cas transcriptional modulation
CN104651399A (zh) 2014-12-31 2015-05-27 广西大学 一种利用CRISPR/Cas系统在猪胚胎细胞中实现基因敲除的方法
WO2016142719A1 (en) 2015-03-12 2016-09-15 Genome Research Limited Biallelic genetic modification
KR20160118987A (ko) 2015-04-01 2016-10-12 한양대학교 산학협력단 LincRNA 삭제를 위한 sgRNA 쌍
WO2016182893A1 (en) 2015-05-08 2016-11-17 Teh Broad Institute Inc. Functional genomics using crispr-cas systems for saturating mutagenesis of non-coding elements, compositions, methods, libraries and applications thereof
CN107849581A (zh) 2015-05-19 2018-03-27 Kws种子欧洲股份公司 用于植物中的特异性核酸编辑的方法和构建体
WO2016184989A1 (en) 2015-05-19 2016-11-24 Kws Saat Se Methods for the in planta transformation of plants and manufacturing processes and products based and obtainable therefrom
WO2016205745A2 (en) 2015-06-18 2016-12-22 The Broad Institute Inc. Cell sorting
US20170198302A1 (en) * 2015-11-17 2017-07-13 The Chinese University Of Hong Kong Methods and systems for targeted gene manipulation
CN105316341A (zh) 2015-12-08 2016-02-10 浙江理工大学 一种LncRNA及其在作为前列腺癌检测标记物或前列腺癌预后复发标记物中的应用
WO2017214460A1 (en) 2016-06-08 2017-12-14 Agilent Technologies, Inc. High specificity genome editing using chemically modified guide rnas
EP3536796A1 (en) 2016-06-17 2019-09-11 Edigene Biotechnology Inc. Gene knockout method
CN107513538A (zh) 2016-06-17 2017-12-26 北京大学 基因敲除方法
CN106637421A (zh) 2016-10-28 2017-05-10 北京大学 双sgRNA文库的构建及其应用于高通量功能性筛选研究的方法
WO2018154027A1 (en) 2017-02-22 2018-08-30 Cancer Research Technology Ltd. Cell labelling, tracking and retrieval
CN107090466A (zh) 2017-04-20 2017-08-25 清华大学 双sgRNA表达质粒及其文库的构建方法
WO2019024081A1 (zh) 2017-08-04 2019-02-07 北京大学 特异性识别甲基化修饰dna碱基的tale rvd及其应用
US20210054030A1 (en) 2017-08-04 2021-02-25 Peking University Tale rvd specifically recognizing dna base modified by methylation and application thereof
WO2019191876A1 (en) 2018-04-02 2019-10-10 Peking University Method for Screening and Identifying Functional lncRNAs
US20210163936A1 (en) 2018-04-02 2021-06-03 Peking University Method for screening and identifying functional lncrnas
WO2020125762A1 (en) 2018-12-20 2020-06-25 Peking University Compositions and methods for highly efficient genetic screening using barcoded guide rna constructs
US20220064633A1 (en) 2018-12-20 2022-03-03 Peking University Compositions and methods for highly efficient genetic screening using barcoded guide rna constructs
WO2020192712A1 (en) 2019-03-26 2020-10-01 Peking University Method for identifying functional elements
US20220186210A1 (en) 2019-03-26 2022-06-16 Peking University Method for identifying functional elements

Non-Patent Citations (183)

* Cited by examiner, † Cited by third party
Title
Adamson, B. et al. (2012). "A Genome-Wide Homologous Recombination Screen Identifies The RNA-Binding Protein RBMX as a Component of the DNA-Damage Response," Nat Cell Biol. 14(3):318-328, 27 pages.
Anders, S. et al. (2010). "Differential Expression Analysis For Sequence Count Data," Genome Biol 11:R106, 12 pages.
Auer, T.O. et al. (2014). "CRISPR/Cas9 and TALEN-Mediated Knock-In Approaches In Zebrafish," Methods, 9 pages.
Ausubel, F. et al. (1987). Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York, TOC, 7 pages.
Billon, P. et al. (Sep. 21, 2017). "CRISPR-Mediated Base Editing Enables Efficient Disruption of Eukaryotic Genes through Induction of STOP Codons," Mol Cell 67:1068-1079.
Black, D.L. (2003). Mechanisms of Alternative Pre-Messenger RNA Splicing. Annual Review of Biochemistry. 72:291-336, 64 pages.
Boch, J. et al. (2010, e-pub. May 10, 2010). "Xanthomonas AvrBs3 Family-Type III Effectors: Discovery and Function," Annu Rev Phytopathol 48:419-436.
Boch, J. et al. (Dec. 11, 2009). "Breaking The Code Of DNA Binding Specificity Of TAL-Type III Effectors," Science 326(5959):1509-1512.
Bogdanove, A.J. et al. (Sep. 30, 2011). "TAL Effectors: Customizable Proteins For DNA Targeting," Science 333(6051):1843-1846.
Bradley, K.A. et al. (Nov. 8, 2001). "Identification Of The Cellular Receptor For Anthrax Toxin," Nature 414:225-229.
Bultmann, S. et al. (2012, e-pub. Mar. 2, 2012). "Targeted Transcriptional Activation of Silent oct4 Pluripotency Gene By Combining Designer TALEs and Inhibition of Epigenetic Modifiers," Nucleic Acids Res 40(12):5368-5377.
Burkard, M.E. et al. (Mar. 15, 2012). "Enabling and Disabling Polo-Like Kinase 1 Inhibition Through Chemical Genetics," ACS Chemical Biology 7:978-981.
Canver, M.C. et al. (2015). "BCL11A Enhancer Dissection By Cas9-Mediated In Situ Saturating Mutagenesis," Nature 527:192-197.
Chapman, J.R. et al. (Aug. 24, 2012). "Playing The End Game: DNA Double-Strand Break Repair Pathway Choice," Mol. Cell. 47:497-510.
Chen, B. et al. (Dec. 19, 2013). "Dynamic Imaging of Genomic Loci In Living Human Cells By an Optimized CRISPR/Cas System," Cell 155:1479-1491.
Chen, D. et al. (2011). "Bortezomib as The First Proteasome Inhibitor Anticancer Drug: Current Status and Future Perspectives," Curr. Cancer Drug Targets 11:239-253.
Chen, F. et al. (Sep. 2011, e-published on Jul. 17, 2011). "High-Frequency Genome Editing Using ssDNA Oligonucleotides With Zinc-Finger Nucleases," Nature Methods 8(9):753-755.
Christian, M. et al. (Oct. 2010, e-pub. Jul. 26, 2010). "Targeting DNA Double-Strand Breaks With TAL Effector Nucleases," Genetics 186(2):757-761.
Clancy, S. (2008). "RNA Splicing: Introns, Exons and Spliceosome," Nature Education 1:31, 3 pages.
Cong, L. et al. (2012). "Comprehensive Interrogation of Natural TALE DNA-Binding Modules and Transcriptional Repressor Domains," Nat Commun. 3:968, 14 pages.
Cong, L. et al. (Feb. 15, 2013). "Multiplex Genome Engineering Using CRISPR/Cas Systems," Science 339(6121):819-823, 9 pages.
Cristea, S. et al. (2013, e-pub. Oct. 5, 2012). "In vivo Cleavage of Transgene Donors Promotes Nuclease-Mediated Targeted Integration," Biotechnol. & Bioeng. 110(3):871-880.
Crooks, G. E. et al. (2004). "WebLogo: A Sequence Logo Generator," Genome Res. 14:1188-1190.
De Wilt, L.H.A.M. et al. (2012, e-pub. Oct. 18, 2011). "Proteasome-Based Mechanisms of Intrinsic and acquired Bortezomib Resistance In Non-Small Cell Lung Cancer," Biochem. Pharmacol. 83:207-217.
Deng, D. et al. (2012). "Recognition of Methylated DNA By TAL Effectors," Cell research 22(10):1502-1504.
Deng, D. et al. (Feb. 10, 2012, e-pub. Jan. 5, 2012). "Structural Basis For Sequence-Specific Recognition Of DNA By TAL Effectors," Science 335(6069):720-723, 9 pages.
Duan, J. et al. (2004). "Structural and Functional Analysis Of Mutations At The Human Hypoxanthine Phosphoribosyl Transferase (HPRT1) Locus," Human Mutation 23:599-611.
Dupuy, A. et al. (Nov. 13, 2013). Targeted Gene Therapy of Xeroderma Pigmentosum Cells Using Meganuclease and TALEN, PLoS One 8(11):e78678, 8 pages.
Engler, C. et al. (May 14, 2009). "Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based On Type IIs Restriction Enzymes," PLoS One 4:e5553, 9 pages.
Engreitz, J.M. et al. (Nov. 17, 2016). "Local Regulation of Gene Expression by IncRNA Promoters, Transcription and Splicing," Nature 539(7629):452-455, with Supplemental, 19 pages.
Extended European Search Report, dated May 10, 2022, for European Patent Application No. 20777188.2, 10 pages.
Extended European Search Report, dated Oct. 20, 2021, for European Patent Application No. 18913494.3, 10 pages.
Ezkurdia, I. et al. (2014, e-pub. Jun. 16, 2014). "Multiple Evidence Strands Suggest That There May Be as Few as 19,000 Human Protein-Coding Genes," Hum. Mol. Genet. 23:5866-5878.
Fang, G. et al. (Dec. 2012). "Genome-Wide Mapping of Methylated Adenine Residues In Pathogenic Escherichia coli Using Single-Molecule Real-Time Sequencing," Nature biotechnology 30(12):1232-1239, 23 pages.
Findlay, G.M. et al. (2014). "Saturation Editing of Genomic Regions By Multiplex Homology-Directed Repair," Nature 513:120-123.
Franke, N.E. et al. (2012, e-pub. Sep. 23, 2011). "Impaired Bortezomib Binding To Mutant β5 Subunit of The Proteasome Is The Underlying Basis For Bortezomib Resistance In Leukemia Cells," Leukemia 26:757-768.
Freshney, R. I., (1987) "Culture of Specific Cell Types" Chapter 20 in Culture of Animal Cells: A Manual of Basic Techniques, Alan R. Liss & Co., New York; pp. 257-260, 270-273.
Fu, S. et al. (Jun. 18, 2010). "The Structure of Tumor Endothelial Marker 8 (TEM8) Extracellular Domain and Implications For Its Receptor Function For Recognizing Anthrax Toxin," PLoS One 5:e11203, 10 pages.
Fu, Y. et al. (May 7, 2015). "N6-Methyldeoxyadenosine Marks Active Transcription Start Sites In Chlamydomonas," Cell 161:1-14.
Fu, Y. et al. (Sep. 2013). "High-Frequency Off-Target Mutagenesis Induced By CRISPR-Cas Nucleases In Human Cells," Nat Biotechnol 31:822-826, 13 pages.
Garg, A. et al. (2012, e-pub. May 11, 2012). "Engineering Synthetic TAL Effectors With Orthogonal Target Sites," Nucleic Acids Res 40(15):7584-7595.
Garst, A.D. et al. (2017, e-pub. Dec. 12, 2016). "Genome-Wide Mapping of Mutations At Single-Nucleotide Resolution For Protein, Metabolic and Genome Engineering," Nat. Biotechnol. 35:48-55.
Genbank NR_110533.1, Shin, S.Y. et al. (Oct. 8, 2016). "Homo sapiens DiGeorge Syndrome Critical Region Gene 5 (Non-Protein Coding) (DGCR5). Transcript Variant 3, Non-Coding RNA," 2 pages.
Gilbert, L.A. et al. (Jul. 18, 2013). "CRISPR-Mediated Modular RNA-Guided Regulation Of Transcription In Eukaryotes," Cell 154:442-451.
Gilbert, L.A. et al. (Oct. 23, 2014). "Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation," Cell 159:647-661.
Goyal, A. et al. (2017, e-pub. Sep. 30, 2016). "Challenges of CRISPR/Cas9 Applications For Long Non-Coding RNA Genes," Nucleic Acids Res 45(3):e12, 13 pages.
Greer, E.L. et al. (May 7, 2015). "DNA Methylation on N6-Adenine in C. elegans," Cell 161:868-878, 26 pages.
Gürlebeck, D. et al. (2006). "Type III Effector Proteins From The Plant Pathogen Xanthomonas and Their Role In The Interaction With The Host Plant," J Plant Physiol. 163(3):233-255.
Guttman, M. et al. (2011). "lincRNAs Act In The Circuitry Controlling Pluripotency and Differentiation," Nature 477(7364):295-300, 32 pages.
Harlow, E. et al. (1988). Antibodies A Laboratory Manual, Table of Contents only, 9 pages.
Hart, T. et al. (2014). "Measuring Error Rates In Genomic Perturbation Screens: Gold Standards For Human Functional Genomics," Molecular Systems Biology 10:733, 16 pages.
Hart, T. et al. (Dec. 3, 2015). "High-Resolution CRISPR Screens Reveal Fitness Genes and Genotype-Specific Cancer Liabilities," Cell 163:1-12.
He, Y.-F. et al. (Sep. 2, 2011). "Tet-Mediated Formation of 5-Carboxylcytosine and Its Excision By TDG In Mammalian DNA," Science 333(6047):1303-1307, 10 pages.
Heckl, D. et al. (Sep. 30, 2014). "Generation of Mouse Models of Myeloid Malignancy With Combinatorial Genetic Lesions Using CRISPR-Cas9 Genome Editing," Nat. Biotechnol. 32(9):941-946, 14 pages.
Heidari, N. et al. (2014). "Genome-Wide Map of Regulatory Interactions In The Human Genome," Genome Res. 24:1905-1917.
Hess, G.T. et al. (2016, e-pub. Oct. 31, 2016). "Directed Evolution Using dCas9-Targeted Somatic Hypermutation In Mammalian Cells," Nat. Methods, 10 pages.
Hsu, P.D. et al. (Sep. 2013). "DNA Targeting Specificity of RNA-Guided Cas9 Nucleases," Nat Biotechnol. 31:827-832, 17 pages.
Hsu, PD. et al. (Jun. 5, 2014). "Development and Applications of CRISPR-Cas9 For Genome Engineering," Cell 157(6):1262-1278, 34 pages.
Hu, J. et al. (2014). "Direct Activation of Human and Mouse Oct4 Genes Using Engineered TALE and Cas9 Transcription Factors," Nucleic Acids Res 42(7):4375-4390, 19 pages.
Huang, Y. et al. (Oct. 2014). "Connections Between TET Proteins and Aberrant DNA Modification In Cancer," Trends Genet 30(10):464-474, 25 pages.
International Preliminary Report on Patentability Opinion, dated Jun. 16, 2021, for PCT Application No. PCT/CN2019/127080, filed Dec. 20, 2019, 4 pages.
International Preliminary Report on Patentability Opinion, dated Sep. 28, 2021, for PCT Application No. PCT/CN2020/081283, filed Mar. 26, 2022, 5 pages.
International Search Report and Written Opinion, dated Jul. 1, 2020, for PCT Application No. PCT/CN2020/081283, filed Mar. 26, 2022, 10 pages.
International Search Report and Written Opinion, dated Mar. 25, 2020, for PCT Application No. PCT/CN2019/127080, filed Dec. 20, 2019, 8 pages.
International Search Report, dated Jan. 4, 2019, for PCT Application No. PCT/CN2018/081635, 6 pages.
International Search Report, dated May 14, 2018, for PCT Application No. PCT/CN2017/096510, English Translation, 2 pages.
International Search Report, dated May 7, 2018, for PCT Application No. PCT/CN2017/095988, English Translation, 3 pages.
Ito, S. et al. (Aug. 26, 2010). "Role of Tet Proteins In 5mC to 5hmC Conversion, ES-Cell Self-Renewal and Inner Cell Mass Specification," Nature 466(7310):1129-1133.
Jackson, S.P. (2002). "Sensing and Repairing DNA Double-Strand Breaks," Carcinogenesis 23(5):687-696.
Jiao, X. et al. (2012). "DAVID-WS: A Stateful Web Service To Facilitate Gene/Protein List Analysis," Bioinformatics 28:1805-1806.
Jinek, M. et al. (Aug. 17, 2012, e-pub. Jun. 28, 2012). "A Programmable Dual-RNA-Guided DNA Endonuclease In Adaptive Bacterial Immunity," Science 337(6096):816-821, 14 pages.
Joung, J. et al. (Aug. 17, 2017). "Genome-Scale Activation Screen Identifies a LncRNA Locus Regulating A Gene Neighborhood," Nature 548(7667):343-346, 28 pages.
Kaufman, R.J. et al. (1987). "Translational Efficiency of Polycistronic mRNAs and Their Utilization To Express Heterologous Genes In Mammalian Cells," The EMBO Journal 6(1):187-195.
Kay, S. et al. (2009). "How Xanthomonas Type III Effectors Manipulate The Host Plant," Curr Opin Microbiol 12(1):37-43.
Kay, S. et al. (Oct. 26, 2007). "A Bacterial Effector Acts As a Plant Transcription Factor and Induces a Cell Size Regulator," Science 318(5850):648-651.
Kim, H. et al. (Nov. 2011). "Surrogate Reporters for Enrichment of Cells With Nuclease-Induced Mutations," Nat. Methods 8(11):941-943.
Kim, H.J. et al. (2009). "Targeted Genome Editing In Human Cells With Zinc Finger Nucleases Constructed Via Modular Assembly," Genome Res. 19:1279-1288.
Kim, Y. et al. (Mar. 2013, e-pub. Feb. 17, 2013). "A Library of TAL effector Nucleases Spanning The Human Genome," Nat Biotechnol 31(3):251-258.
Kim, Y.-G. et al. (Feb. 1996). "Hybrid Restriction Enzymes: Zinc Finger Fusions To Fok I Cleavage Domain," Proc. Natl. Acad. Sci. USA 93:1156-1160.
Kohli, R.M. et al. (Oct. 24, 2013). TET Enzymes, TDG and The Dynamics of DNA Demethylation, Nature 502(7472):472-479, 21 pages.
Koike-Yusa, H. et al. (2014, e-pub. Dec. 23, 2013). "Genome-wide Recessive Genetic Screening In Mammalian Cells With a Lentiviral CRISPR-Guide RNA Library." Nat Biotechnol 32:267-273.
Kolde, R. et al. (2012, e-pub. Jan. 12, 2012). "Robust Rank Aggregation For Gene List Integration and Meta-Analysis," Bioinformatics 28(4):573-580.
Konermann, S. et al. (Jan. 29, 2015). "Genome-Scale Transcriptional Activation By an Engineered CRISPR-Cas9 Complex," Nature 517:583-588.
Koziol, M.J. et al. (Jan. 2016). "Identification of Methylated Deoxyadenosines In Vertebrates Reveals Diversity In DNA Modifications," Nature Structural & Molecular Biology 23(1):24-30, 23 pages.
Kretz, M. et al. (Jan. 10, 2013). "Control of Somatic Tissue Differentiation By The Long Non-Coding RNA TINCR," Nature 493:231-235, 16 pages.
Kriaucionis, S. et al. (Apr. 16, 2009). The Nuclear DNA Base 5-Hydroxymethylcytosine Is Present In Purkinje Neurons and The Brain, Science 324(5929):929-930.
Kubik, G. et al. (2014). "Programmable and Highly Resolved In Vitro Detection of 5-Methylcytosine By TALEs," Angew Chem Int Ed Engl 53(23):6002-6006.
Kubik, G. et al. (2015). "Achieving Single-Nucleotide Resolution of 5-Methylcytosine Detection With TALEs," Chembiochem 16(2):228-231.
Kubik, G. et al. (Jan. 5, 2015). "Programmable Sensors of 5-Hydroxymethylcytosine," J Am Chem Soc 137(1):2-5.
Lackner, D.H. et al. (Dec. 17, 2015). "A Generic Strategy For CRISPR-Cas9-Mediated Gene Tagging," Nature Communications 6:10237, 7 pages.
Leng, N. et al. (2013, e-pub. Feb. 21, 2013). "EBSeq: An Empirical Bayes Hierarchical Model For Inference in RNA-Seq Experiments," Bioinformatics 29(8):1035-1043.
Li, B. et al. (2011). "RSEM: Accurate Transcript Quantification From RNA-Seq Data With Or Without a Reference Genome," BMC Bioinformatics 12:323, 16 pages.
Li, H.L. et al. (2016, e-pub. Oct. 23, 2015). "Efficient Genomic Correction Methods In Human iPS Cells Using CRISPR-Cas9 System," Methods 101:27-35.
Li, K. et al. (Aug. 28, 2014). "Optimization of Genome Engineering Approaches with the CRISPR/Cas9 System," PLoS One 9(8):e105779, 10 pages.
Li, W. et al. (2014). "MAGeCK Enables Robust Identification Of Essential Genes From Genome-Scale CRISPR/Cas9 Knockout Screens," Genome Biol 15:554, 12 pages.
Liao, S. et al. (2015, e-pub. Jun. 29, 2015). "Enriching CRISPR-Cas9 Targeted Cells By Co-Targeting The HPRT Gene," Nucleic Acids Res. 43(20):e134, 8 pages.
Lim, K.H. et al. (Jul. 5, 2011). "Using Positional Distribution To Identify Splicing Elements and Predict Pre-mRNA Processing Defects In Human Genes," Proc. Natl. Acad. Sci. USA. 108(27):11093-11098.
Lin, N. et al. (Mar. 2014). "An Evolutionarily Conserved Long Noncoding RNA TUNA Controls Pluripotency and Neural Lineage Commitment," Mol. Cell. 53:1005-1019.
Liu, S.J. et al. (Jan. 6, 2017). "CRISPRi-Based Genome-Scale Identification of Functional Long Noncoding RNA Loci In Human Cells," Science 355(6320):1-19.
Liu, Y. et al. (Dec. 2018, e-pub. Nov. 5, 2018). "Genome-Wide Screening For Functional Long Noncoding RNAs In Human Cells By Cas9 Targeting Of Splice Sites," Nature Biotechnology 36(12):1203-2010.
Long, C. et al. (Jan. 31, 2018). "Correction of Diverse Muscular Dystrophy Mutations in Human Engineered Heart Muscle By Single-Site Genome Editing," Science Advances 4:eaap9004, 11 pages.
Lü, S. et al. (2013). "The Resistance Mechanisms of Proteasome Inhibitor Bortezomi," Biomarker Research 1:13, 9 pages.
Lyras, D. et al. (2009). "Toxin B Is Essential For Virulence of Clostridium difficile," Nature 458:1176-1179.
Ma, H. et al. (Mar. 10, 2015). "Multicolor CRISPR Labeling of Chromosomal Loci In Human Cells," Proc. Natl. Acad. Sci. USA 112:3002-3007.
Maiti, A. et al. (Oct. 14, 2011). "Thymine DNA Glycosylase Can Rapidly Excise 5-Formylcytosine and 5-Carboxylcytosine: Potential Implications For Active Demethylation of CpG Sites," J Biol Chem 286(41):35334-35338.
Mak, A.N.-S. et al. (Feb. 10, 2012) "The Crystal Structure of TAL Effector PthXo1 Bound To Its DNA Target," Science 335(6069):716-719, 11 pages.
Mali, P. et al. (Feb. 15, 2013, e-pub. Jan. 3, 2013). "RNA Guided Human Genome Engineering via Cas9," Science 339(6121):823-826, 8 pages.
Matlin, AJ. et al. (May 2005). "Understanding Alternative Splicing: Towards a Cellular Code," Nat. Rev. Mol. Cell Biol. 6:386-398.
Maurer, S. et al. (Nov. 4, 2016). "Interrogating Key Positions of Size-Reduced TALE Repeats Reveals a Programmable Sensor of 5-Carboxylcytosine," ACS Chem Biol 11(12):3294-3299.
Meyers, R.M. et al. (2017, e-pub. Oct. 30, 2017). "Computational Correction Of Copy Number Effect Improves Specificity Of CRISPR-Cas9 Essentiality Screens In Cancer Cells," Nat Genet 49:1779-1784.
Michlits, G. et al. (2017, e-pub. Oct. 16, 2017). "CRISPR-UMI: Single-Cell Lineage Tracing Of Pooled CRISPR-Cas9 Screens," Nat Methods 14:1191-1197.
Miller, J.C. et al. (Feb. 2011, e-pub. Dec. 22, 2010). "A Tale Nuclease Architecture For Efficient Genome Editing," Nature Biotechnology 29(2):143-148.
Miller, J.C. et al. (May 2015, e-pub. Mar. 23, 2015). "Improved Specificity of TALE-Based Genome Editing Using An Expanded RVD Repertoire," Nat Methods 12(5):465-471.
Mitamura, T. et al. (Oct. 24, 1997). "Structure-Function Analysis of The Diphtheria Toxin Receptor Toxin Binding Site By Site-Directed Mutagenesis," J. Biol. Chem. 272:27084-27090.
Miyaoka, Y. et al. (Mar. 2014). "Isolation of Single-Base Genome-Edited Human iPS Cells Without Antibiotic Selection," Nat Methods 11:291-293, 15 pages.
Morbitzer, R. (Dec. 14, 2010). "Regulation of Selected Genome Loci Using De Novo-Engineered Transcription Activator-Like Effector (TALE)-Type Transcription Factors," Proc Natl Acad Sci USA 107(50):21617-21622.
Moscou, M.J. et al. (Dec. 11, 2009, e-pub. Oct. 29, 2009). "A Simple Cipher Governs DNA Recognition By TAL Effectors," Science 326:1501, 1 page.
Muller, R.Y. et al. (2015). "An Efficient Method for Electroporation of Small Interfering RNAs into ENCODE Project Tier 1 GM12878 and K562 Cell Lines," J. Biomol. Tech 26:142-149.
Murugan, R.N. et al. (Sep. 30, 2011). "Plk1-Targeted Small Molecule Inhibitors: Molecular Basis For Their Potency and Specificity," Mol. Cells 32:209-220.
Mussolino, C. et al. (2011, e-pub. Aug. 3, 2011). "A Novel TALE Nuclease Scaffold Enables High Genome Editing Activity In Combination With Low Toxicity," Nucleic Acids Res 39(21):9283-9293.
Nakade, S. et al. (Nov. 20, 2014). "Microhomology-Mediated End-Joining-Dependent Integration of Donor DNA In Cells And Animals Using TALENs and CRISPR/Cas9," Nature Communications 5:5560, 8 pages.
Ng, B, et al. (Dec. 2004). "Increased Noncanonical Splicing of Autoantigen Transcripts Provides The Structural Basis For Expression of Untolerized Epitopes," Journal of Allergy and Clinical Immunology 114(6):1463-1470, 17 pages.
Nishimasu, H. et al. (Feb. 27, 2014). "Crystal Structure of cas9 in Complex With Guide RNA and Target DNA," Cell 156:935-949.
O'Brien, A. et al. (2014). "GT-Scan: Identifying Unique Genomic Targets," Bioinformatics 30(18):2673-2675.
Orlando, S.J. et al. (Aug. 2010, e-pub, Jun. 8, 2010). "Zinc-finger Nuclease-Driven Targeted Integration Into Mammalian Genomes Using Donors With Limited Chromosomal Homology," Nucleic Acids Research 38(15):e152, 15 pages.
Pastor, W.A. et al. (Jun. 2013). "TETonic Shift: Biological Roles of TET Proteins In DNA Demethylation and Transcription," Nat Rev Mol. Cell Biol. 14(6):341-356, 34 pages.
Peng, J. et al. (2015). "High-Throughput Screens In Mammalian Cells Using The CRISPR-Cas9 System," FEBS J. 282:2089-2096.
Perez, E.E. et al. (2008, e-pub. Jun. 29, 2008). "Establishment of HIV-1 Resistance In CD4+ T Cells By Genome Editing Using Zinc-Finger Nucleases," Nat. Biotechnol. 26:808-816.
Pfaffeneder, T. et al. (2011). "The Discovery of 5-Formylcytosine In Embryonic Stem Cell DNA," Angew Chem Int Ed Engl 50(31):7008-7012.
Phillips, E.R. et al. (2007). "DNA Double-Strand Break Repair and Development," Oncogene 26:7799-7808.
Potter, C.J. et al. (Apr. 13, 2010). "Splinkerette PCR For Mapping Transposable Elements In Drosophila," PLoS One 5(4):e10168, 9 pages.
Qian, L. et al. (May 2014, e-pub. Mar. 25, 2014). "Bidirectional Effect Of Wnt Signaling Antagonist DKK1 On The Modulation Of Anthrax Toxin Uptake," Science China Life Sciences 57(5):469-481.
Quinn, J.J. et al. (Jan. 2016). "Unique Features of Long Non-Coding RNA Biogenesis and Function," Nat. Rev. Genet. 17:47-62.
Ramakrishna, S. et al. (Feb. 26, 2014). "Surrogate Reporter-Based Enrichment of Cells Containing RNA-Guided Cas9 Nuclease-Induced Mutations," Nature Communications 5:3378, 10 pages.
Ratel, D. et al. (2006). "N6-Methyladenine: The Other Methylated Base of DNA," BioEssays 28:309-315.
Rathi, P. et al. (Jul. 18, 2016). "Isolation of Human Genomic DNA Sequences With Expanded Nucleobase Selectivity," J Am Chem Soc 138(31):9910-9918.
Ren, Q. et al. (Mar. 9, 2015). "A Dual-Reporter System for Real-Time Monitoring and High-throughput CRISPR/Cas9 Library Screening of the Hepatitis C Virus," Scientific Reports 5:8865.
Richardson, C.D. et al. (2016, e-pub. Jan. 20, 2016). "Enhancing Homology-Directed Genome Editing By Catalytically Active and Inactive CRISPR-Cas9 Using Asymmetric Donor DNA," Nat. Biotechnol., 7 pages.
Rinn, J.L. et al. (2012). "Genome Regulation By Long Noncoding RNAs," Annu. Rev. Biochem 81:145-166, 25 pages.
Robinson, M.D. et al. (2008, e-pub. Aug. 29, 2007). "Small-Sample Estimation Of Negative Binomial Dispersion, With Applications To SAGE Data," Biostatistics 9:321-332.
Sakuma, T. et al. (2016, e-pub. Dec. 17, 2015). "MMEJ-Assisted Gene Knock-In Using TALENs and CRISPR-Cas9 With The PITCh Systems," Nat. Protoc. 11(1):118-133.
Sambrook, J. et al. (1989). Molecular Cloning: A Laboratory Manual, 3rd Ed. 29 pages.
Sambrook, J. et al. (2001). Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 3rd ed., 1 page, Table of Contents.
Sanjana, N. (Mar. 19, 2014). "Human and Mouse Non-Targeting Guides From GeCKOV2 Library," SanjanaLab, 5 pages.
Savić, N. et al. (2016). "Advances In Therapeutic CRISPR/Cas9 Genome Editing," Translational Research: The Journal Of Laboratory and Clinical Medicine 168:15-21.
Schmid-Burgk. J.L. et al. (Jul. 28, 2016). "CRISPaint Allows Modular Base-Specific Gene Tagging Using a Ligase-4-Dependent Mechanism," Nature Communications 7(12338):1-12.
Schmierer, B. et al. (2017, e-pub. Oct. 9, 2017). "CRISPR/Cas9 Screening Using Unique Molecular Identifiers," Molecular Systems Biology 13:945, 8 pages.
Seed, B. (1987). "An LFA-3 cDNA Encodes a Phospholipid-Linked Membrane Protein Homologous To Its Receptor CD2," Nature 329:840-842.
Shalem, O. et al. (Jan. 3, 2014). "Genome-Scale CRISPR-Cas9 Knockout Screening In Human Cells," Science 343:84-87.
Shechner, D.M. et al. (2015, e-pub. Jun. 1, 2015). "Multiplexable, Locus-Specific Targeting Of Long RNAs With CRISPR-Display," Nat Methods 12:664-670.
Steegmaier, M. et al. (Feb. 20, 2007). "BI 2536, A Potent and Selective Inhibitor of Polo-Like Kinase 1, Inhibits Tumor Growth In Vivo," Curr. Biol. 17:316-322.
Suzuki, E. et al. (Dec. 22, 2011). "Molecular Mechanisms of Bortezomib Resistant Adenocarcinoma Cells," PLoS One 6(12):e27996, 11 pages.
Taggart, A.J. et al. (2012). "Large-Scale Mapping of Branchpoints In Human Pre-mRNA Transcripts In Vivo," Nat. Struct. Mol. Biol. 19(7):719-721, 9 pages.
Tahiliani, M. et al. (May 15, 2009). "Conversion of 5-Methylcytosine To 5-Hydroxymethylcytosine In Mammalian DNA By MLL Partner TET1," Science 324(5929):930-935, 11 pages.
Tan, Y.Y. et al. (Aug. 15, 1989). "In Vitro Evaluation Of 6-Thioguanine and Alpha-Interferon as a Therapeutic Combination In HL-60 and Natural Killer Cells," Cancer Res. 49:4431-4434.
Tao, L. et al. (2016). "Frizzled Proteins Are Colonic Epithelial Receptors For C. difficile Toxin B," Nature 538:350-355.
Tsuji, S. et al. (2016). "Sequence-Specific Recognition of Methylated DNA by an Engineered Transcription Activator-Like Effector Protein," Chem. Commun. 52:14238-14241.
Uren, A.G. et al. (2009, e-pub. Apr. 30, 2009). "A High-Throughput Splinkerette-PCR Method For The Isolation and Sequencing of Retroviral Insertion Sites," Nat. Protoc. 4(5):789-798.
Valton, J. et al. (Nov. 9, 2012). "Overcoming Transcription Activator-Like Effector (TALE) DNA Binding Domain Sensitivity To Cytosine Methylation," J Biol Chem 287(46):38427-38432.
Van Overbeek, M. et al. (Aug. 18, 2016). "DNA Repair Profiling Reveals Nonrandom Outcomes at Cas9-Mediated Breaks," Mol. Cell 63:1-14.
Wacker, S.A. et al. (Mar. 2012). "Using Transcriptome Sequencing To Identify Mechanisms Of Drug Action and Resistance," Nat. Chem. Biol. 8:235-237.
Wang, T. et al. (Jan. 3, 2014). "Genetic Screens In Human Cells Using The CRISPR-Cas9 System," Science 343:80-84.
Wang, T. et al. (Nov. 27, 2015). "Identification and Characterization of Essential Genes In The Human Genome," Science 350(6264):1096-1101.
Warf, M.B. et al. (2010). "Role of RNA Structure In Regulating Pre-mRNA Splicing," Trends Biochem. Sci. 35:169-178, 21 pages.
Wei, W. et al. (Mar. 24, 2006). "The LDL Receptor-Related Protein LRP6 Mediates Internalization and Lethality Of Anthrax Toxin," Cell 124:1141-1154.
Wion, D. (Mar. 2006). "N6-Methyl-Adenine: An Epigenetic Signal For DNA-Protein Interactions," Nature Reviews Microbiology 4:183-192.
Wu, H. et al. (Sep. 2015). "Charting Oxidized Methylcytosines At Base Resolution," Nat Struct Mol Biol 22(9):656-661, 14 pages.
Xu, H. et al. (2015). "Sequence Determinants of Improved CRISPR sgRNA Design," Genome Res. 25:1147-1157.
Yang, J. et al. (2016). "Assembly of Customized TAL Effectors Through Advanced ULtiMATE System." TALENs: Methods and Protocols pp. 49-60.
Yang, J. et al. (May 2014) Complete Decoding of TAL Effectors For DNA Recognition, Cell Research 24(5):628-631.
Yang, J. et al. (Sep. 27, 2013). "ULtiMATE System For Rapid Assembly of Customized TAL Effectors," PLoS One 8(9):e75649, 8 pages.
Yin, B. et al. (Jul. 2007). "PCR-Based Procedures To Isolate Insertion Sites of DNA Elements," Biotechniques 43(1):79-84.
Yu, C. et al. (Feb. 5, 2015). "Small Molecules Enhance CRISPR Genome Editing in Pluripotent Stem Cells," Cell Stem Cell 16:142-147.
Yu, M. et al. (Jun. 8, 2012). "Base-Resolution Analysis of 5-Hydroxymethylcytosine In The Mammalian Genome," Cell 149(6):1368-1380, 23 pages.
Yuan, P. et al. (2015, e-pub. Dec. 30, 2014). "Chondroitin Sulfate Proteoglycan 4 Functions As The Cellular Receptor For Clostridium difficile Toxin B," Cell Res 25:157-168.
Zhang, G. et al. (May 7, 2015). "N6-Methyladenine DNA Modification In Drosophila," Cell 161:1-14.
Zhang, Y. et al. (2017, e-pub. Oct. 12, 2017). "Deciphering TAL Effectors For 5-Methylcytosine and 5-Hydroxymethylcytosine Recognition," Nature Communications, 8(1):1-9.
Zhou, Y. et al. (2016). "Simultaneous Generation of Multi-Gene Knockouts in Human Cells," FEBS Letters 590:4343-4353.
Zhou, Y. et al. (2017, e-pub. Jan. 13, 2017). "Painting a Specific Chromosome With CRISPR/Cas9 For Live-Cell Imaging," Cell Res. 27:298-301, 4 pages.
Zhou, Y. et al. (May 22, 2014). "High-Throughput Screening Of a CRISPR/Cas9 Library For Functional Genomics In Human Cells," Nature 509:487-491.
Zhou, Yuexin, Hongmin Zhang, and Wensheng Wei. "Simultaneous generation of multi-gene knockouts in human cells." FEBS letters 590.23 (2016): 4343-4353. (Year: 2016). *
Zhu, S. et al. (2017). "Genome-Wide CRISPR/Cas9 Screening for High-Throughput Functional Genomics in Human Cells," Methods Mol Biol 1656:175-181.
Zhu, S. et al. (Dec. 2016, e-pub. Oct. 31, 2016). "Genome-Scale Deletion Screening Of Human Long Non-Coding RNAs Using a Paired-Guide RNA CRISPR-Cas9 Library," Nature Biotechnology 34(12):1279-1286.

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